Melatonin down-regulates volume-sensitive chloride channels in fibroblasts
Ismail Ben Soussia & Frédérique Mies & Robert Naeije &
Vadim Shlyonsky
Received: 14 May 2012 /Revised: 16 July 2012 /Accepted: 17 July 2012 /Published online: 27 July 2012 # Springer-Verlag 2012
Abstract Melatonin has been reported to present with vaso- relaxant and anti-fibrotic properties. We hypothesized that melatonin may down-regulate volume-regulated anion channels (VRAC) in fibroblasts to limit their migration and proliferation. While acute exposure of L929 fibroblasts to melatonin did not result in a significant decrease in VRAC current, pretreatment with 100 μM melatonin for 1 h decreased swelling-dependent activation of anion currents by 83 % as measured by whole-cell perforated patch-clamp technique. This down-regulation of VRAC currents was dose-dependent with a half-maximal inhibition of 3.02±0.48 μM. Overnight treatment of cells with 100 nM melatonin had the same inhibitory potency as a 1- h treatment with 100 μM. A similar down-regulatory effect of melatonin on VRAC was observed in primary rat lung fibroblasts. The effect of melatonin was prevented by luzindole and K185 that suggests implication of MT2 re- ceptor. GF109203X, a protein kinase C inhibitor, blocked melatonin’s action on VRAC, indicating that MT2 receptor activation results in stimulation of PKC. Consequently, mel- atonin inhibited regulatory volume decrease following hy- potonic swelling of cells. Melatonin also decreased the migration of L929 fibroblasts through the same pathways that blocked VRAC. There was no significant inhibition of cell proliferation. Our study suggests that the attenuation of fibrosis and vascular remodeling by melatonin seen in ani- mal models of hypertension and pulmonary fibrosis might be, in part, related to blunted fibroblast migration possibly through protein kinase C-mediated decrease in chloride channel activity.
Keywords Patch-clamp . Anion channel . Melatonin receptor . Cell volume . Cell migration . Cell proliferation
Introduction
The pathophysiology of lung fibrosis remains incompletely understood [20]. Lung fibrosis is often complicated by pulmonary hypertension that may arise from vascular remodeling due to over-expression of cytokines and growth factors [31]. Tissue remodeling seen in these conditions originates from an abnormal activity of several cell types including fibroblasts that have substantial migratory and proliferative capacity and secrete growth factors [12, 23, 38]. The physiology of fibroblasts is influenced by many different signaling pathways. We have recently reported that volume-regulated anion channels (VRAC) in adventitial pulmonary fibroblasts were inhibited by bone morphogenet- ic protein 2 in a protein kinase C-dependent manner and that this inhibition was associated with decreased migration and differentiation of these cells [34]. Other studies reported sensitivity of fibroblasts to serotonin, ET-1, TGFβ, reactive oxygen species, and melatonin [9, 19, 41, 43].
Melatonin is a hormone secreted by the pineal gland. Several studies suggest that it has an anti-fibrotic effect. In a model of N-nitro-L-arginine-methyl ester-induced hyperten- sion in rats, it prevented the development of left ventricular fibrosis [30]. Melatonin also significantly decreased the fi- brotic score (evaluated by Aschoft’s criteria and lung hy- droxyproline content) in bleomycin-induced pulmonary fibrosis [48]. On the other hand, pinealectomy in rats caused hypertension within 2 weeks of the operation as well as
I. Ben Soussia : F. Mies : R. Naeije : V. Shlyonsky (*) Department of Physiology, Université Libre de Bruxelles, Route de Lennik 808, CP604,
1070 Brussels, Belgium
e-mail: [email protected]
vascular remodeling in the kidneys as evidenced by thicken- ing of the vascular wall, narrowing of arteriolar lumen, and adventitial fibrosis [50]. Injection of melatonin following pinealectomy corrected this hypertension [17]. Many other
studies described a vasorelaxant and antihypertensive effect of melatonin in animal models [4, 13, 16, 22, 28, 35, 40]. None of these studies, however, explored the cellular and molecular mechanisms involved in the action of melatonin.
The goal of the present study was to examine if the beneficial effect of melatonin in reducing fibrosis and vas- cular remodeling could be attributed in part to changes in the chloride permeability of the fibroblasts. We report here that melatonin modifies the cell volume regulatory machinery of L929 fibroblasts and primary pulmonary fibroblasts in culture. Melatonin inhibits regulatory volume decrease y4following osmotic swelling by down-regulating VRAC in these cells. We also show that this inhibition is dose- dependent, requires fixation of melatonin to its receptor, and involves activation of protein kinase C. Finally, we show that melatonin inhibits migration of these cells through the same pathways that inhibit VRAC.
Materials and methods
Reagents
All reagents including melatonin, inhibitors, and cell culture media components were purchased from Sigma-Aldrich (Leuven, Belgium).
Cell culture
L929 fibroblasts were purchased from the European Collection of Cell Cultures through Sigma-Aldrich and cultured in DMEM media supplemented with 10 % fetal calf serum, 1 % penicillin-streptomycin in a humidified incubator at 37 °C and 5 % CO2. Primary rat lung fibroblasts were pur- chased from Cell Applications (San Diego, CA, USA) and cultured in RFGM medium (Rat Fibroblast Growth Medium, R116-500, Cell Applications) in a humidified incubator at 37 °C and 5 % CO2. Cells were passaged using 0.25 % trypsin-EDTA after reaching a maximal confluence of 80 %. The L929 cells were used for up to six consecutive passages, and primary rat lung fibroblasts were used up to the passage 5.
Electrophysiology
Nystatin-perforated whole-cell patch clamp was used to record volume-regulated anion currents. Briefly, cells were plated on glass coverslips coated with 100 μg/cm2 of gelatin and used between 12 and 36 h after plating. Coverslips were placed in the patch-clamp chamber and continuously per- fused with a standard physiological solution containing (in millimoles per liter) the following: 140 NaCl, 4 KCl, 10 HEPES, 1 CaCl2, 1 MgCl2, and 10 D-glucose, titrated to pH 7.3 with NaOH (final [Na] 0 144 mM). Osmolarity was 305
mOsmol/L. In hypotonic solution, 40 mmol/L of NaCl were omitted, and the osmolarity of the solution measured by freezing point osmometer was 230 mOsm. For whole-cell measurements, the pipette solution contained (in millimoles per liter) the following: NaCl 10, KCl 10, K-aspartate 130, HEPES 10, D-glucose 11, and 400 μg/mL nystatin, titrated to pH 7.2 with KOH (final [K] 0 134 mM). Osmolarity was 295 mOsm/L. Stock solution of nystatin (40 mg/mL in Me2SO) was prepared daily. In experiments involving var- iations of Cl gradient across cell membrane, NaCl in the bath solution and/or K-aspartate in the pipette solution were replaced by Na-aspartate and KCl, respectively, as indicated in the figure legend. In an experiment studying effect of tetraethylammonium (TEA+), 5 mM of NaCl in hypotonic bath solution was replaced by 5 mM of TEA-Cl. The patch pipettes were double-step-pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany) using a vertical puller (PC-10, Narishige International, London, UK). Filled pipettes had resistances of 4–6 MΩ. Whole-cell patch-clamp configuration induced by nystatin permeabilization was achieved within 15–20 min at room temperature. Access resistance (Rs < 30 mOhms) was stable for at least 20 min after stabilization, and it was compensated by 70 %. Cells were used within 60 min after being taken from the incuba- tor. The holding potential was -50 mV. Voltage ramp pro- tocols consisted of a step to -100 mV (200 ms in duration) followed by a ramp ranging from -100 to +100 mVover 1 s. Ramps were applied every 14 s. Values of whole-cell cur- rents were obtained by points averaging on the I/V curve between +90 and +95 mV. Voltage steps protocol consisted of voltage steps from -100 to +100 mV (20-mV intervals) and 1.5-s duration applied from a holding potential of
-50 mV. The VRAC current represents the difference in whole-cell current density at +90 mV in hypotonic and isotonic bath solutions. Due to appreciable decline in VRAC activity after each cell culture passage, the experimental design included a series of patch-clamp experiments done in parallel on treated and control cells from the same pas- sage. Mean cell capacitance of L929 cells was 23.1±1.4 pF and that of primary rat lung fibroblasts was 75.3±16.8 pF.
PKC activity
PKC activity was measured using the PepTag® assay according to manufacturer notice (Promega, Leiden, Nether- derland). Fifteen million control or treated cells were ho- mogenized in 0.75 mL of PKC extraction buffer containing (in millimoles per liter) 25 Tris–HCl (pH 7.4), 0.5 EDTA, 0.5 EGTA, 10 β-mercaptoethanol, and protease inhibitor cocktail (Roche) followed by a 30-s sonication to disinte- grate the cell membranes. After centrifugation at 10,000× g for 5 min, the pellet was discarded, and the supernatant was ultracentrifugated at 100,000× g for 1 h to separate the
membranes from the cytosol. The membrane fraction was resuspended in 100 μl of PKC extraction buffer supplemented with 0.05 % of Triton X-100 and was sonicated for 30 s. Protein concentration was measured by the Bradford method (Pierce) and was readjusted to the same value in all samples using PKC extraction buffer. PKC activity was assayed in a reaction mix- ture containing (in millimoles per liter) the following: 20 HEPES (pH 7.4), 1.3 CaCl2, 1 DTT, 10 MgCl2, and 1 ATP as well as 5 μg of phosphatidylserine and 2 μg of PepTag® C1 peptide for 30 min at 30 °C. Purified bovine PKC enzyme provided in the kit served a positive control. The reaction was stopped by heating in 95 °C for 10 min, and phosphorylated and non-phosphorylated PepTag® fluorescent peptides were separated by electrophoresis on agarose gel (0.8 % in 50 mmol/L Tris–HCl, pH 8.0). Gels were photographed under UV light, and the optical density of bands was measured using standard analysis plugins of ImageJ software (version 1.42q).
Cell migration
The migration of L929 fibroblasts was studied using the Oris Cell Migration Assay (Tebu-Bio, Boechout, Belgium). Eighty thousand cells in serum-containing media were seeded per well in 96-well plate, into which cell-seeding stoppers made from a medical-grade silicone were inserted to restrict cell seeding to the outer annular regions of the well. After cell attachment, seeding stoppers were removed, and wells were washed with serum-free media to remove non-attached cells. Cells were labeled using 2 μM Calcein- AM for 15 min, and ×10 microscopic fluorescent photo of each well was taken using a Scion charge-coupled device camera and MicroManager program. Control and treated cells were then allowed to migrate into the central area for 15 h. In order to prevent cell proliferation, 5 μg/mL of mitomycin was added to the media. Fluorescence microsco- py photos of wells with migrated cells were again taken. The percent surface in the central area occupied by cells before and after cell migration was evaluated by using CellProfiler program [18]. Data are expressed as a percentage of gap closure in the central region. Each experimental condition was repeated with the cells from at least three different passages, and a minimum of five wells were seeded for each assay condition.
Cell volume measurements
Mean cell volume was measured in cell suspensions by electronic cell sizing (Scepter handheld cell counter, Milli- pore) using a 60-μm aperture. Cells in subconfluent cultures were harvested with minimal trypsin (0.05 %), suspended in
gentle agitation for 15 min at 37 °C. Hypotonic media (0.67× isotonic) was established by mixing one volume of cell suspension with two volumes of 0.5× PBS. Mean cell volume was taken from the peak of the Gaussian distribu- tion on the cell volume histograms measured for total cells in 100 μl of suspension. The regulatory volume decrease, i.e., percent of recovery from the initial swelling (%RVD) was calculated using the following formula: %RVD 0 100 × (Vt2 - Vt30)/(Vt2 - Vt0), where Vt0, Vt2, and Vt30 are mean cell volumes at time 0, 2, and 30 min, respectively. Changes in values were expressed as relative cell volume normalized to the value in isotonic solution (at time 0).
Cell proliferation
Cell proliferation was assessed using the Cell Counting Kit- 8 (CCK-8, Sigma-Aldrich, Leuven, Belgium). This colori- metric assay consists in the use of water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitro- phenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt]. The amount of yellow-colored formazan dye, a reduc- tion product of WST-8 generated by the activity of dehy- drogenases in cells is directly proportional to the number of living cells. Two thousand cells per well were grown in three separate 96-well plates. Cells were allowed to attach in the wells overnight and then were treated with vehicle, melatonin, or DIDS every 24 h onwards. After 24, 48, or 72 h, 10 μl of the CCK-8 solution were added in each well of one of three plates, and the plate was further incubated for 4 h. The absorbance was measured at 450 nm using a microplate reader.
Statistical analysis
Data are represented as mean ± SEM. Student’s non-paired t test and analysis of variance were used for comparisons, and differences with p values <0.05 were considered significant.
Results
VRAC become activated, and thus their activity becomes measurable only after an osmotic challenge. Therefore, all patch-clamp recordings were carried on L929 fibroblasts exposed to a hypotonic solution (230 mOsm/L). The current density in isotonic solution averaged 2.71±0.35 pA/pF and rose to a value of 21.80±2.66 pA/pF (n 0 17) within a few minutes of exposure to hypotonic solution (Fig. 1a, b). These hypotonicity-activated currents were not significantly prevented by potassium channel blocker TEA+ (Fig. 1c, d,
phosphate buffered solution containing Ca2+ and Mg2+ g; p >0.05) but were inhibited by dihydro-4,4′-diisothiocya-
(PBS) and centrifuged for 5 min at 300× g. Cell were resus- pended in PBS at 2–3×105 cells/mL and incubated with
nostilbene-2,2′-disulfonic acid (DIDS), a non-specific blocker of anion channels. In this case, recordings in the
Fig. 1 Hypotonicity stimulates whole-cell chloride current in L929 cells. Continuous record- ings in a control cell (a), in a cell receiving tetraethylammo- nium TEA (c), DIDS (e), TEA + DIDS (g), and hypotonic perfusion without calcium (i) are shown. A voltage ramp sig- nal (from -100 to +100 over
1 s) was applied every 14 s. Between ramps, cells were held at -50 mV. Bars indicate the
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isotonic solution revealed a current of 3.83±0.86 pA/pF, and the application of hypotonic solution supplemented with 100 μM DIDS increased this current to a value of only 7.71±1.07 pA/pF (Fig. 1e, f). Simultaneous application of DIDS and TEA in hypotonic solution completely prevented activation of the current and even decreased it below basal level in some instances (Fig. 1g, h). A summary of these experiments is presented in Fig. 2a. The DIDS-sensitive component represents 80 % of hypotonicity-activated cur- rent, while the remaining 20 % is represented by K currents. We next sought to determine whether removal of Ca2+ ions from the hypotonic solution affects hypotonicity-activated currents. While the kinetics of whole-cell current activation was slower (Fig. 1i), no significant difference in current amplitude was observed under this condition (Fig. 2b).
The hypotonicity-induced currents slowly inactivated at the voltages positive to +60 mV. This is demonstrated in Fig. 2c that shows families of membrane currents recorded during 1.5-s steps to potentials between -100 and +100 mV (20-mV step increments) under control conditions and after switching to hypotonic solution. These slowly inactivating currents completely disappear in the presence of DIDS (Fig. 2d). Additional evidence that hypotonic solution main- ly activates anion cell conductance comes from the measure- ments of the reversal potential of the current-to-voltage curve of hypotonicity-activated current. Figure 2e shows representative records of hypotonicity-induced currents (baseline current subtracted) in three different chloride gra- dient conditions. Summary of reversal potentials shown in Fig. 2f demonstrates that experimentally measured reversal potential of the current follows theoretical Nernst potential for chloride anion under different experimental conditions.
In summary, our results demonstrate that anion transport is a dominating component of hypotonicity-activated current. For simplicity, hypotonicity-induced current is referred to as a volume-regulated anion channel current (VRAC current).
Acute exposure of cells to melatonin in hypotonic solu- tion did not result in a significant decrease in VRAC current (p >0.05, n 0 9). However, when cells were pre-incubated with 100 μM melatonin for 30–60 min at 37 °C before patching, the response to hypotonicity was blunted (Figs. 3a, b vs. 1a, b). Small currents activated by hypotonic solution in melatonin-pretreated cells still showed some voltage inactivation (Fig. 3c, d). VRAC currents decreased from 19.09±2.67 pA/pF in 17 control cells to 3.70± 0.93 pA/pF in eight cells pretreated with melatonin (Fig. 2e, p <0.001). Melatonin failed to further inhibit VRAC currents in the presence of DIDS; in DIDS- perfused cells, VRAC current averaged 2.36±0.67 pA/pF (n 0 5) vs. 2.18±0.83 pA/pF in five melatonin-treated and perfused with DIDS cells (Fig. 3e, p >0.05). We conclude that melatonin down-regulates DIDS-sensitive VRAC cur- rent. The effect of melatonin on VRAC was dose-dependent
with a half-maximal inhibition of 3.02±0.48 μM (Hill’s coefficient of 0.53, Fig. 3f). A similar maximal inhibitory effect was observed when cells were exposed overnight to 100 nM of melatonin (Fig. 3e).
Since melatonin action might be receptor-mediated or receptor-independent, we first tested the effect of luzindole, a non-selective antagonist of melatonin receptors, on melatonin-inhibited VRAC current. In cells exposed to 10 μM of luzindole for 30 min before the application of melatonin, the inhibitory effect of melatonin was attenuated (Fig. 4a). VRAC current that averaged 27.68±3.01 pA/pF (n 0 5 cells) decreased to a value of 8.02±1.83 pA/pF in cells treated with melatonin (p 0 0.005 vs. control, n 0 5 cells) and were restored to 20.90±2.57 pA/pF in cells pretreated with luzindole before melatonin administration (p 0 0.003 vs. melatonin, p 0 0.12 vs. control, n 0 5). Similar results were obtained with K185, a selective antagonist of the melatonin receptor 2. The effect of melatonin on VRAC current inhi- bition was completely suppressed in the presence of 1 nM K185 applied 10 min before melatonin (Fig. 4b). VRAC currents decreased from a value of 21.51±4.49 pA/pF in control cells (n 0 8 cells) to 3.31±1.33 pA/pF in the presence of melatonin (p 0 0,002 vs. control, n 0 6) and increased back to 20.91±3.90 pA/pF when we pretreated cells with K185 (n 0 6 cells, p 0 0.92 vs control, p 0 0.001 vs. melatonin). These results indicate that the effect of melatonin on VRAC activity occurs largely via the activation of melatonin receptors and most likely through the MT2 receptor.
The activation of MT2 receptor is associated with a PLC/
PKC signaling pathway [15, 51], and we have shown pre- viously that PKC inhibits VRAC in pulmonary fibroblasts [34]. To investigate the implication of PKC, cells were pre- treated with the PKC inhibitor GF109203X at a concentra- tion of 2.5 μM for 30 min before application of melatonin. Under these conditions, the effect of melatonin completely disappeared (Fig. 4c). In control cells, the VRAC current was 27.68±3.01 pA/pF (n 0 5). It fell to 8.02±1.83 pA/pF in the presence of melatonin (n 0 5, p 0 0.0005 vs. control) and increased back to 26.52±3.55 pA/pF with pretreatment with GF109203X (n 0 5 cells, p 0 0.8 vs. control, p 0 0.002 vs melatonin).
Independent evidence that melatonin activates PKC in a receptor-dependent manner comes from the direct PKC activ- ity measurements in L929 cells using the PepTag assay. The phosphorylation of C1 peptide, a specific PKC substrate, was tested in presence of 4 μg of purified membrane protein. Figure 4d shows a representative agarose gel of phosphorylat- ed and non-phosphorylated PepTag® C1 peptides whose ratio reflects PKC activity. PKC activity increased by 144 % in cells treated with melatonin (p 0 0.002 vs. control), and this stimu- lation was inhibited in the presence of K185 (p 0 0.04 vs. melatonin). K185 alone did not affect PKC activity (p >0.05 vs. control).
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Fig. 2 Summary of hypotonicity-activated chloride current character- ization. a Summary of TEA, DIDS, and TEA + DIDS effects on hypotonicity-activated currents. Mean ± SE differences between the whole-cell current under the hypotonic and control perfusion that averaged between +90 and +95 mV are shown. The effect of TEA was not significant, p >0.05, n 0 6, while DIDS significantly inhibited hypotonicity-activated current; triple asterisks p <0.001, n 0 12 vs. con- trol (n 0 17). The presence of TEA and DIDS together in the hypotonic solution completely prevents hypotonicity-activated current; section sign p <0.001 vs. control and p 0 0.018 vs. DIDS, n 0 6. b Ca-free hypotonic solution does not affect hypotonicity-activated current. Mean ± SE differences between the whole-cell current under the hypotonic and control perfusion that averaged between +90 and +95 mV are shown; p >0.05, n 0 5 vs. control (n 0 6). c, d Voltage dependence of the current induced by hypotonic solutions. Voltage steps from -100 to +100 mV (20-mV intervals) and 1.5-s duration
were applied from a holding potential of -50 mV. Before exposure to hypotonic solutions, only small time-independent currents were ob- served (top traces). In hypotonic solution (bottom traces), larger cur- rents could be recorded that slowly inactivate at large positive potentials. These voltage-inactivated currents disappear in the presence of DIDS. e, f Dependence of reversal potential of hypotonicity- activated currents (baseline current subtracted) on Cl- gradient across cell membrane. Three different conditions with cation gradients remaining the same were used: 108 mM bath/20 mM pipette (n 0 12), 108 mM bath/108 mM pipette (n 0 5), and 20 mM bath/108 mM pipette (n 0 7). e Representative I/V curves of the three Cl- gradient conditions showed in f. The curve of experimental reversal potential as a function of chloride equilibrium Nernst potential (C) shows a slope value of 0.83±0.02. Dotted line represents theoretical curve for ideally selective Cl channel
Fig. 3 Melatonin inhibits hypotonicity-activated whole- cell currents. Continuous recordings in a melatonin-treated cell (a). A voltage ramp signal (from -100 to +100 over 1 s) was applied every 14 s. Between ramps, cells were held at
-50 mV. Bar indicates the dura- tion of the hypotonic perfusion. b I/V curves as an expansion of the record shown in a. c, d Volt- age dependence of the
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We next examined if the effect of melatonin on VRAC current was reproduced in primary cultures of rat lung fibroblasts. The results demonstrate that 1-h treatment with 100 μM melatonin induced down-regulation of VRAC sim- ilar to L929 cells (Fig. 5). In control cells, the VRAC current was 6.96±1.52 pA/pF (n 0 7) which was decreased to 0.44± 0.20 pA/pF in the presence of melatonin (n 0 6, p 0 0.006 vs. control).
Inhibition of volume-sensitive anion channels by mela- tonin in L929 cells implies that melatonin may affect their cell volume regulation. Regulatory volume decrease follow- ing osmotic swelling in hypotonic solution was investigated (Fig. 6a, b). Non-treated cells showed a 61.6±4.2 %RVD, while in cells pretreated with 100 μM melatonin, this value
was only 40.6±4.6 % (n 0 5, p 0 0.01), which represents 34 % inhibition. When cells were challenged by DIDS- containing hypotonic solution, they showed a 32.4±3.8
%RVD (n 0 3, p 0 0.003 vs. control). In isotonic solution, cells pretreated with melatonin had slightly higher volumes (1.99±0.04 vs. 1.86±0.06 pL in control cells); however, this 7 % difference did not reach statistically significant level (p >0.05, n 0 6). We conclude that strong down- regulation of VRAC in L929 results only in modest inhibition of cell volume regulation.
We have shown previously that down-regulation of VRAC activity affects the migration of pulmonary fibro- blasts in culture [34]. Given the inhibitory action of mela- tonin on VRAC currents, we tested the effect of melatonin
Fig. 4 Inhibition of VRAC requires fixation of melatonin to MT2 receptor and involves activation of PKC. a Non-specific melatonin receptor antagonist luzindole attenuates effect of melatonin. Cells were treated with 10 μM luzindole for 30 min before melatonin administra- tion; double asterisks p 0 0.0005 vs. control (n 0 5), number sign p 0 0.003 vs. melatonin (n 0 5) and p 0 0.12 vs. control (n 0 5). b MT2 receptor antagonist K185 prevents effect of melatonin. Cells were treated with 1 nM K185 for 10 min before melatonin; double asterisks p 0 0.002 vs. control (n 0 6), number sign p 0 0.001 vs. melatonin (n 0 6), and p 0 0.92 vs. control (n 0 5). c Effect of melatonin on VRAC current requires activation of PKC. The pretreatment with the specific inhibitor of PKC, GF109203X (GFX) at 2.5 μM for 15 min before melatonin prevents the effect of melatonin; triple asterisks p 0 0.0005 vs. control (n 0 5), number sign p 0 0.002 vs. melatonin (n 0 5) and p 0 0.8 vs.
control (n 0 5). d Effect of melatonin on PKC activity. Representative 0.8 % agarose gel with PepTag® C1 peptide and analysis of four experiments are shown. Prior to densitometry analysis, background in gels was removed using ImageJ software. PKC activity was calculated as a ratio of band optical density between phosphorylated and non- phosphorylated peptide (migrating downwards and upwards, respec- tively) and expressed as a relative activity normalized to control cells. Lanes on the gel correspond to experimental conditions seen below in the bar graph. PMA, phorbolemyristate acetate served a positive con- trrol for PKC activation and C+ and C- lanes represent internal positive (10 ng of purified bovide PKC enzyme) and negative (with no protein) control of the assay; double asterisks p 0 0.002 vs. control; number sign p 0 0.04 vs. melatonin alone; and single asterisk p 0 0.01 vs. control
on cell migration. Figure 7a shows that DIDS, a blocker of VRAC, inhibits L929 cell migration. The percentage of gap closure following 15 h of cell migration decreased from 9.57±0.21 % in control cells to 3.28±0.87 % in cells that were allowed to migrate in the presence of 100 μM DIDS (n 0 4, p 0 0.0004 vs. control). In the presence of 100 μM melatonin, this migration index averaged 4.57±0.60 % (n 0 4, p 0 0.002 vs. control). When added together with DIDS, melatonin did not further inhibit cell migration (Fig. 7a). These results indicate that the inhibition of cell migration by melatonin is a consequence of VRAC down-regulation. We next explored whether the signaling pathways involved in melatonin inhibition of cell migration were also inhibiting VRAC. Figure 7b, c shows that the effect of melatonin on cell migration was suppressed in the presence of both luzin- dole and K185. Furthermore, the effect of melatonin on cell
migration required an active PKC pathway since GF109203X counteracted the observed inhibition of cell migration by melatonin. Migration in control cells was 10.11±0.70 %, 3.80±0.62 % in the presence of melatonin (Fig. 7d, n 0 3, p 0 0.002 vs. control), and 10.88±0.57 % when exposed to GF109203X before melatonin application (p 0 0.45 vs. control, p 0 0.001 vs. melatonin). These results suggest that melatonin inhibits cell migration through the same pathways that inhibit VRAC.
VRAC may also control cell proliferation [46]. We sought to determine whether VRAC inhibition by melatonin also resulted in a decreased cell proliferation. Figure 8 demonstrates that there was a trend to decreased cell prolif- eration at 72 h with both treatment, 100 μM melatonin and 100 μM DIDS, but this effect did not reach statistical significance (n 0 6, p >0.05 vs. control).
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Fig. 5 Melatonin prevents hypotonicity-activated chloride current in primary rat lung fibroblasts. Continuous recordings in a control (a) and in 100 μM melatonin-treated cell (c). A voltage ramp signal (from
-100 to +100 over 1 s) was applied every 14 s. Between ramps, cells were held at -50 mV. Bars indicate the duration of the hypotonic perfusion. b, d I/V curves as an expansion of the records shown in a
and c, respectively. e Summary of melatonin effects on IVRAC in rat lung fibroblasts. Mean ± SE differences between the whole-cell current under the hypotonic and control perfusion that averaged between +90 and +95 mV are shown; double asterisks p 0 0.006 (n 0 6) vs. control (n 0 7)
Discussion
In this report, several lines of evidence demonstrate that hypotonicity-induced whole-cell currents in fibroblasts are mostly carried by the chloride channels. These include slow voltage inactivation of the current, sensitivity to DIDS, as well as a strong dependence on chloride ion gradient across the cell membrane. Melatonin significantly influences cell
volume regulation in the vascular fibroblast cell line L929, as well as in primary rat lung fibroblasts, by affecting VRAC activity, which in turn modulates cell migration. We also show that overnight treatment of cells with 100 nM melatonin, a concentration slightly above its phys- iological plasma level, has the same strong down-regulatory action on VRAC as that of 1-h treatment with 100 μM melatonin. In this regard, it is important to note that daily
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Fig. 6 Melatonin affects osmotic cell volume regulation of L929 cells.
aRegulatory volume decrease following hypotonic swelling is expressed as relative cell volume normalized to the value in isotonic solution (at time 0). b Percent of recovery from initial swelling
(%RVD) in control cells, melatonin-treated cells, and in the presence of 500 μM DIDS. Double asterisks p 0 0.003 vs. control (n 0 5), single asterisk p 0 0.01 vs. control (n 0 3). c Mean cell volume in control cells and cells treated with 100 μM melatonin for 1 h
Fig. 7 L929 cell migration. Examples of cell culture well images showing cell migration between 0 and 15 h in control and melatonin-treated cells are shown. a–d Data represent a percent of gap closure in the central area of cell culture wells after 15 h of cell migration. a DIDS (100 μM) and melatonin (100 μM) inhibit cell migration; triple asterisks p <0.001 vs. control (n 0 4). Melatonin does not further inhibit cell migration in the presence of DIDS; sec- tion sign p 0 0.0003 vs. control, not significant vs. DIDS alone.
bMelatonin binds to its recep- tor to affect L929 cell migra- tion. Luzindole at 10 μM prevents the effect of melato- nin; single asterisk p 0 0.01 vs. control, number sign p 0 0.02 vs. melatonin and p >0.05 vs. control (n 0 3). c The selective antagonist of MT2 receptor K185 at 1 nM inhibits the effect of melatonin on cell migration; double asterisks p 0 0.008 vs. control, number sign p 0 0.01 vs. melatonin and p >0.05 vs. control (n 0 4). d Melatonin
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activates PKC to inhibit L929 cell migration; double asterisks p 0 0.002 vs. control, number sign p 0 0.001 vs. melatonin and p >0.05 vs. control (n 0 3)
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pulmonary fibrosis [41, 48, 50]. It is also important to note that melatonin loses its activity in chronic hypoxia models of pulmonary hypertension [13], which could possibly, in part, account for adventitial remodeling seen under these conditions [12, 23].
We did not observe a significant inhibition of cell prolif- eration induced by melatonin or by DIDS. We conclude that inhibition of VRAC by melatonin and DIDS in L929 cells might be compensated by other ion transporters involved in cell volume regulation. This conclusion is supported by the
Fig. 8 VRAC do not participate significantly in proliferation of L929 cells. Data represent relative cell numbers normalized to the control value at 24 h. After 72 h, we observed a non-significant decline in the numbers of viable cells both in the presence of 100 μM melatonin or 100 μM DIDS compared to control cells (p >0.05, n 0 7)
production of melatonin in humans and animals does not exceed 50 μg, while bioavailability of exogenous melatonin remains very low and, in particular, doses of 100 mg/kg were necessary to prevent hypertension and vascular remodeling in pinealectomized rats [17].
Volume-regulated anion channels participate in the mod- ulation of cell membrane potential, cell proliferation, cell differentiation, and cell migration [10, 42, 46, 49]. Our results show that melatonin inhibits the activity of VRAC and that this inhibition is dose-dependent and receptor- mediated. Two mammalian melatonin receptor subtypes have been identified by molecular cloning studies. The MT1 (or Mel1A or MTNR1A) and MT2 (or Mel1B or MTNR1B) receptor subtypes are present in humans and other mammals and are a G-protein-coupled receptors. It was shown that melatonin receptor 2 (MT2) rather than MT1 is the preferential actor in the antihypertensive [1, 2, 13, 14, 45] and cardiovascular protective effects of melato- nin [21, 32]. Our results indicate that, indeed, the activation of MT2 receptor is responsible for the decreased migration of fibroblasts due to inhibition of VRAC by melatonin. Moreover, we show that the downstream signaling cascade following activation of MT2 receptor involves stimulation of protein kinase C, which is inhibited by GF109203F. These observations are in agreement with that of previous studies showing that PKC down-regulates VRAC [27, 34]
and also confirm that melatonin activates the PKC pathway [3, 6, 15, 36, 51].
VRAC might be the rate-limiting step in determination of cell migration [33], while excessive cell migration might cause fibrosis and vascular remodeling [31, 37]. Our obser- vations of the inhibition of fibroblast migration by melato- nin are in agreement with several previous reports [8, 11, 24, 29]. We propose that decreased fibroblast migration in the presence of melatonin might partially explain an anti- fibrotic effect of melatonin seen in animal models of
observation that strong inhibition of VRAC results only in modest suppression of regulatory volume decrease follow- ing osmotic challenge (see Figs. 1 and 4). We suggest that this compensation is sufficient in case of volume changes during cell proliferation, while cell volume changes associated with cell migration would require all cell volume regulation transporters including VRAC.
It is tempting to speculate that any cell expressing MT2 receptors would respond to elevated concentrations of mel- atonin by a perturbation in cell volume regulation. In this regard, cycling changes in cell volume have been described in pinealocytes [44], which themselves express MT2 recep- tors. Cell volume increased during the periods of melatonin secretion [44]. We also observed a slight swelling of L929 fibroblasts following melatonin treatment although this ef- fect was not statistically significant. Nonetheless, cyclic pinealocyte swelling as a result of autocrine melatonin inhi- bition of VRAC in the pineal gland deserves to be more closely investigated.
VRAC and Ca-activated chloride channels participate in the regulation of cell membrane voltage in several cell types [10]. Pharmacological inhibition of VRAC currents led to a repolarization of cell membrane voltage following cell de- polarization resulting from VRAC activation by hydrogen peroxide in β-pancreatic cells [10]. Sustained inhibition of volume-sensitive channels in smooth muscle cells by mela- tonin could possibly explain the preventive action of mela- tonin on vasospastic effect of H2O2 in endothelium-denuded segments of human umbilical artery [28]. In fibroblasts, reported values of resting membrane potential vary from low values between -10 and -25 mV [5, 25] to higher values in excess of -50 mV [7, 39] recorded in a variety of cells grown in culture. Moreover, spontaneous endoge- nous potential oscillations were reported in fibroblasts, which spanned the range between -25 and -50 mV [26]. Since the intracellular chloride concentration in fibroblasts appears to be approximately 20–25 mM [47] while that of interstitial compartment is accepted to be 98–105 mM, physiologically relevant equilibrium potential for chloride ions situates between -35 and -42 mV. If we also assume a contribution of bicarbonate ions that permeate through VRAC, overall equilibrium potential for anions shifts closer to 0 mV. These considerations imply that the inhibition of
chloride channel activity by melatonin may potentially have opposing effects on resting membrane voltage in fibroblasts depending on the direction of electrochemical chloride gradient. Although we did not measure resting membrane potential in L929 and primary lung fibroblast cells, our data suggest that electrochemical chloride gradient appears to be outward in these cells, and the down-regulation of VRAC results in the accumulation of negative changes in the cell, most likely in hyperpolarization-induced decrease in intracellular calcium and, consequently, in inhibition of cell migration.
In summary, our study suggests that down-regulation of VRAC by melatonin leads to a decreased fibroblast migra- tory capacity. This mechanism could contribute to the preventive effect of melatonin on tissue remodeling seen in animal models of hypertension and pulmonary fibrosis. Thus, our results are of potential interest to the development of specific therapies for patients with idiopathic lung fibrosis or pulmonary arterial hypertension.
Acknowledgments This study was supported by funds from the Funds National pour la Recherche Scientifique (to R.N.) and the Funds d’Encouragement à la Recherche of Université Libre de Bruxelles (to V.S.). We would like to express our gratitude to Sarah Sariban-Sohraby for invaluable comments on the manuscript.
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