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Am J Physiol Cell Physiol 290: C1000-C1008, 2006. First published November 2, 2005; doi:10.1152/ajpcell.00353.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells

Moon Young Kim,1 Guo Hua Liang,1 Ji Aee Kim,1 Young Ju Kim,2 Seikwan Oh,3 and Suk Hyo Suh1

1Department of Physiology, 2Department of Obstetrics and Gynecology, and 3Department of Neuroscience, Medical Research Institute, College of Medicine, Ewha Woman's University, Seoul, Republic of Korea

Submitted 14 July 2005 ; accepted in final form 30 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca2+-activated K+ (BKCa) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca2+]i. [Ca2+]i and membrane potentials were strongly correlated. In whole cell clamped cells, BKCa currents were activated by increasing [Ca2+]i via cell dialysis with pipette solution, and the activated BKCa currents were further enhanced by S1P. When [Ca2+]i was buffered at 1 µM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BKCa channel activity in a reversible manner and shifted the relationship between Ca2+ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; 1 mM) using a patch pipette, GDPbetaS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BKCa current and channel activation. These results suggest that S1P enhances BKCa channel activity by increasing Ca2+ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca2+ influx through Ca2+ entry channels. Inasmuch as S1P activates BKCa channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca2+ mobilization in human endothelial cells.

sphingolipid metabolites; intracellular second messenger; Ca2+ mobilization

SPHINGOSINE-1-PHOSPHATE (S1P), which is released by activated platelets, elicits various responses in vascular tissues, including angiogenesis, endothelial cell migration, survival, and proliferation (10, 22), and endothelial nitric oxide (NO) synthase activation (15). S1P can increase intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells and thereby induces important biological responses, given that intracellular Ca2+ in endothelial cells controls many essential cellular functions, which range from a rapid NO secretory response to longer-term responses such as cell growth and death. S1P-induced increase in [Ca2+]i in endothelial cells might be due to Ca2+ release from intracellular stores (25) and/or to the activation of Ca2+ entry channels such as nonselective cation channel (NSC) (26) and/or Ca2+ release-activated Ca2+ channels (CRAC) (25).

S1P recently was shown to bind cell surface S1P receptors. These receptors were initially called endothelial differentiation gene (Edg) receptors and have been renamed S1P receptors. Five subtypes of these receptors have been identified: S1P1 (Edg-1), S1P2 (Edg-5), S1P3 (Edg-3), S1P4 (Edg-6), and S1P5 (Edg-8) (35). S1P receptors are members of G protein-coupled receptors (21), and many actions of S1P are mediated via these receptors. S1P1 and S1P3 activation regulates angiogenesis and stimulates wound healing (8, 17). In contrast, S1P2 activation inhibits migration of melanoma cells (39). In addition, S1P activates various ion channels via these G protein-coupled receptors. S1P activates NSC in a GTP-dependent manner via a pertussis toxin-sensitive G protein (26) and Cl– channel (37).

Alternatively, S1P also may directly regulate various biological processes as a second messenger. S1P releases Ca2+ from intracellular Ca2+ stores independently of inositol trisphosphate (24) and stimulates signal transduction pathways, including Ca2+ mobilization and phospholipase D activation, independently of S1P receptor expression (36). S1P may act as a second messenger to evoke store-operated Ca2+ entry independently of G proteins (18). Furthermore, S1P has been implicated as a second messenger in cellular proliferation, cell survival, and apoptosis suppression (34). S1P may control apoptosis via extracellular signal-regulated kinase activation (7) and c-Jun NH2-terminal kinase inhibition (5). However, the intracellular targets of S1P remain much more elusive, and direct modulation of ion channels by S1P has not yet been reported.

In the present study, we examined the Ca2+-mobilizing properties of S1P in human endothelial cells and present evidence that it activates Ca2+-activated K+ (BKCa) channels via a mechanism independent of G protein-coupled receptors. BKCa channel activation may increase Ca2+ driving force by hyperpolarizing membrane and thereby modulating [Ca2+]i in human endothelial cells.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Human umbilical vein endothelial cell culture. This study was approved by the Institutional Review Board for Human Research at Ewha Woman's University in Seoul. We used primary cultured human umbilical vein endothelial cells (HUVECs). HUVECs were isolated from human umbilical cord veins from normal pregnancies in nonsmoking women (n = 25) by collagenase treatment immediately after delivery, as previously described (19, 38). HUVECs were grown in DMEM containing 20% FCS plus 10% hypoxanthine-aminopterin-thymidine (HAT) 50x supplement (Life Technologies). HUVECs were identified as endothelial cells in origin by their cobblestone appearance at confluence and positive staining with acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, MA) and mouse anti-human factor VIII (Calbiochem, San Diego, CA).

Cell culture was maintained at 37°C in fully humidified air with 5% CO2 atmosphere. The cells were then detached by exposure to trypsin, reseeded on gelatin-coated coverslips, and maintained in culture for 2–4 days before use. Measurements were performed on nonconfluent cells.

Electrophysiology. Membrane potentials were monitored in current-clamp mode with an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) using a nystatin-perforated patch (100 mg/ml). Whole cell currents were measured using ruptured patches. Voltages were monitored in voltage-clamp mode using an EPC-9 amplifier. A holding potential of 0 mV was used for the whole cell experiment. A voltage from –100 to +100 mV of 650-ms duration was applied every 10 s. Currents were recorded at a sampling rate of 1–4 kHz. Inside-out voltage clamps were performed using glass electrodes with tip resistances of ~5–10 M{Omega}. Data were filtered at 1 kHz and stored in a computer for analysis using standard software (Axoscope version 8.2; Axon Instruments, Foster City, CA).

Ca2+ measurements. Cells were loaded with fura-2 AM, and [Ca2+]i was measured in isolated single cells by using a microfluorimeter consisting of an inverted microscope (Leica DM IRB) and a PTI power illuminator system (Photon Technology International). Fura-2 AM (2 µM) was added to the bath, and the cells were incubated for 25 min at 37°C. The cells were then illuminated alternatively at wavelengths of 340 and 380 nm through a chopper wheel (frequency = 50 Hz). Fluorescence was measured at 510 nm, and the signals were corrected for autofluorescence. Free Ca2+ concentrations were calculated from the ratios of fluorescence signals emitted at each excitation wavelength. The calibration procedure was conducted as described previously (29).

Solutions. The standard external solution contained (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 with NaOH. The osmolarity of this solution, as measured with a vapor pressure osmometer (Fiske), was 320 ± 5 mosM. The standard pipette solution contained (in mM) 40 KCl, 100 K-aspartate, 1 MgCl2, 0.1 EGTA, 4 Na2ATP, 10 HEPES, and 5 EGTA, pH 7.2 with KOH (290 mosM). In inside-out mode, the standard external solution was used as the pipette solution and the bathing solution contained (in mM) 150 KCl, 2 MgCl2, and 10 HEPES, pH 7.2 with KOH. For buffering free Ca2+, the appropriate amount of Ca2+ (calculated using CaBuf software, G. Droogmans, Leuven, Belgium; ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) was added in the presence of 5 mM EGTA.

Chemicals. Guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), iberiotoxin (IbTx), and pertussis toxin (PTX) were purchased from Sigma. Fura-2 AM was purchased from Molecular Probes, nystatin was obtained from ICN Biomedicals, and S1P was obtained from Avanti. Nystatin was applied from a stock solution in DMSO. S1P was applied from a stock solution in methanol. The final concentrations of DMSO and methanol were <0.05%.

All experiments were performed at room temperature. Pooled data are presented as means ± SE, and significant differences were detected using Student's t-test (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of S1P on [Ca2+]i and membrane potential. We monitored the changes in [Ca2+]i together with the membrane potentials in HUVECs to examine the effect of S1P on [Ca2+]i and the membrane potentials (Fig. 1). In current-clamped HUVECs, S1P increased [Ca2+]i and hyperpolarized the membrane simultaneously in a reversible manner (Fig. 1A), and there was a strong correlation between [Ca2+]i and the membrane potentials (Fig. 1B). This S1P-induced hyperpolarization was inhibited by IbTx (data not shown), indicating that large-conductance Ca2+-activated K+ (BKCa) current activation might be responsible for S1P-induced hyperpolarization. We thus examined whether S1P activated BKCa currents. In voltage-clamped HUVECs, in which [Ca2+]i was not buffered (0.1 mM EGTA), S1P activated IbTx-sensitive and outwardly rectifying currents (Fig. 1C). The corresponding current-volume (I-V) curves of the currents in the presence of S1P showed a typical BKCa current phenotype, i.e., outward rectification and increased noise at positive potentials (Fig. 1D). Thus IbTx-sensitive and outwardly rectifying currents represent currents through BKCa channels.


Figure 1
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  		Fig. 1. Membrane hyperpolarization and iberiotoxin (IbTx)-sensitive outwardly rectifying current activation by sphingosine-1-phosphate (S1P). A: in nystatin-perforated, current-clamped human umbilical vein endothelial cells (HUVECs), S1P simultaneously increased intracellular Ca2+ concentration ([Ca2+]i) and hyperpolarized the membrane. B: relationship between the membrane potential (Vm) and [Ca2+]i during period a in A. C and D: time course of the membrane current measured at –50 and 100 mV (C) and the current-voltage (I-V) relationship obtained from the voltage ramps labeled 1, 2, and 3 in C (D).

 
BKCa current activation by S1P might be due to an increased [Ca2+]i, because S1P increased [Ca2+]i in endothelial cells (Fig. 1A). In addition, S1P may activate BKCa current via a Ca2+-independent sensitization of BKCa channels. Therefore, we examined whether S1P affects BKCa current in cells when [Ca2+]i was buffered.

S1P modulates BKCa channels. In whole cell clamped cells, loading cells with a Ca2+ solution buffered at various concentrations with the use of a patch pipette activated a similar IbTx-sensitive and outwardly rectifying currents (Fig. 2). The amplitudes of the outwardly rectifying currents were correlated with [Ca2+]i levels (Fig. 2E) and therefore represented BKCa currents. S1P application under these buffered [Ca2+]i conditions further enhanced outward currents without increasing inward currents (Fig. 2, A–C), and the IbTx-sensitive component of the outward current was significantly increased by S1P (Fig. 2B). When [Ca2+]i was buffered at 0.5 (Fig. 2B), 1 (Fig. 3), and 2 µM (Fig. 2C), the outward current was significantly enhanced by S1P (Fig. 2E), indicating a Ca2+-independent sensitization of the BKCa channels by S1P. In contrast, when BKCa current was maximally activated by loading cells with a Ca2+ solution buffered at 5 µM with a patch pipette, it was not further activated by S1P (Fig. 2D). S1P shifted the [Ca2+]i-current relationship to the left without a change in the slope of the curve (Fig. 2E): the [Ca2+]i required to evoke half-maximal activation was changed from 814 ± 40 nM (in the absence of S1P) to 517 ± 66 nM (in the presence of 1 µM S1P), whereas the Hill coefficient was not changed (2.6 ± 0.3 in the absence or presence of S1P) (Fig. 2E).


Figure 2
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  		Fig. 2. Large-conductance, Ca2+-activated K+ (BKCa) current activation and its modulation by S1P in whole cell mode. BKCa currents were activated by loading [Ca2+]i at 0.2 (A), 0.5 (B), 2 (C), or 5 µM (D). A-1, B-1, C-1, and D-1: data points were obtained at 100 and –50 mV during repetitive ramps. A-2, B-2, C-2, and D-2: I-V curves were obtained from the experiments described in A-1, B-1, C-1, and D-1. E: [Ca2+]i-current relationship. Current densities at 100 mV were plotted against [Ca2+]i, and the data obtained were fitted to the Hill equation (solid line). BKCa currents were significantly increased by S1P. *P < 0.05. **P < 0.01. ***P < 0.001.

 

Figure 3
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  		Fig. 3. Concentration-dependent activation of BKCa currents by S1P. BKCa currents were activated by loading [Ca2+]i at 1 µM. A: data points were obtained at 100 and –50 mV during repetitive ramps. B and C: I-V curves were obtained from the experiment described in A. D: difference currents observed before and after S1P application. This I-V relationship was obtained by subtracting the membrane currents in the absence of S1P (1 and 3 in A) from those in its presence (2 and 4 in A), respectively. E: summarized data describing the concentration-response relationship for the S1P effect on BKCa currents. The increases in the current densities induced by S1P were normalized to the current densities activated by loading [Ca2+]i at 1 µM. The normalized increases at 100 mV were plotted against S1P concentration, and the data obtained were fitted to the Hill equation (solid line).

 
The concentration-response relationship for S1P is shown in Fig. 3. BKCa currents were activated by loading cells with a Ca2+ solution buffered at 1 µM, and then S1P was applied. S1P application under this buffered [Ca2+]i condition further enhanced the activated BKCa currents in a reversible and concentration-dependent manner (Fig. 3), but no difference in current enhancement was observed after S1P was added at concentrations between 1 and 5 µM (Fig. 3, D and E), indicating that 1 µM is close to the maximal concentration of S1P with respect to BKCa current activation. The S1P concentration required to evoke half-maximal activation was 403 ± 13 nM, and the Hill coefficient was 2.9 ± 0.2 (Fig. 3E).

BKCa channel activities were observed in inside-out patches. They were activated in a Ca2+- and voltage-dependent manner with a single-channel conductance of 203 ± 13 pS in a 150 mM Na+-containing pipette solution (data not shown). The effect of S1P on BKCa channels was then examined. BKCa channels were activated by clamping free Ca2+ concentration in the bathing solution ([Ca2+]b) at 1 µM (Fig. 4), and S1P was then applied in the bathing solution. On S1P application, BKCa channel activity was increased with a delay of 9.7 ± 0.15 s (n = 11; Fig. 4A), but current amplitudes were unchanged (Fig. 4, A, C, and D). S1P significantly increased mean open time and open probability from 6.5 ± 0.4 ms and 0.177 ± 0.063 to 17.9 ± 1.7 ms and 0.698 ± 0.108, respectively (n = 7; P < 0.001; Fig. 4D). Increased mean open time and open probability were reduced to 7.9 ± 1.5 ms and 0.193 ± 0.084 with a delay of 5.7 ± 0.9 s (n = 9), respectively, after washout (Fig. 4, A–C). The [Ca2+]b-mean open probability relationship at 20 mV is shown in Fig. 5. S1P (1 µM) shifted this relationship to the left without a change in the slope: the EC50 value fell from 1.510 ± 0.082 (in the absence of S1P) to 0.452 ± 0.023 µM (in the presence of S1P), whereas the Hill coefficient remained unchanged at 2.7 ± 0.3. Similarly to S1P, sphingosine also activated BKCa channels in inside-out patches (Fig. 6), whereas S1P did not activate BKCa channels in Ca2+-free bath solution (data not shown).


Figure 4
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  		Fig. 4. S1P-induced BKCa channel activation in inside-out patches. A: BKCa channels were activated by 1 µM Ca2+, and the channel activity (NPo) was further increased by S1P. B: NPo, which was calculated every 51.2 ms (512-point sweeps), is plotted against time. C and D: current traces (C) recorded at the periods labeled a, b, c, and d in B, and their corresponding histograms (D). 01 and 02 indicate the opening level of 1 and 2 channels. NPo, single-channel current amplitudes (i), and mean open times (To) during periods a–d are given.

 

Figure 5
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  		Fig. 5. Relationship between BKCa channel open probability (N x Po) and free Ca2+ concentration in bath solution ([Ca2+]b) at a holding potential of 20 mV, where N is the number of channels in the patch. The data were fitted using the Hill equation. Open probability of BKCa channels at various [Ca2+]b was significantly increased by S1P. **P < 0.01. ***P < 0.001.

 

Figure 6
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  		Fig. 6. Sphingosine-induced BKCa channel activation in inside-out patches. A: BKCa channels were activated by 1 µM Ca2+, and the channel activity was further increased by sphingosine. B: NPo, which was calculated every 51.2 ms (512-point sweeps), is plotted against time. C and D: current traces recorded at periods labeled a, b, c, and d in B, and their corresponding histograms (D). 01 and 02 indicate the opening level of 1 and 2 channels. NPo, i, and To during periods a–d are given.

 
S1P activates BKCa channels independently of G protein. The dependence on GTP of S1P-induced BKCa current and channel activation was examined using the G protein inhibitors GDPbetaS and PTX. In the whole cell configuration, BKCa currents were activated by loading cells with a Ca2+ solution buffered at 0.5 µM, and the effect of the G protein inhibitor GDPbetaS (1 mM) was analyzed by adding it to the patch pipette solution (Fig. 7, A–D). In cells internally perfused with GDPbetaS (Fig. 7, A and B) or treated with PTX for 15 h (data not shown), BKCa currents were further enhanced by S1P (1 µM) application. Furthermore, a combination of GDPbetaS and PTX also did not inhibit S1P-induced BKCa current activation. In cells that were treated with PTX for 15 h and then loaded with GDPbetaS, S1P-induced BKCa current activation also was observed (Fig. 7, C and D).


Figure 7
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  		Fig. 7. G protein-independent activation of BKCa currents by S1P in whole cell mode. BKCa currents were activated by loading [Ca2+]i at 0.5 µM, and cells were internally perfused with 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). A and C: time course of the membrane current measured at –50 and 100 mV. B and D: I-V relationship obtained from voltage ramps labeled 1, 2, 3, and 4 in A and C. In C and D, HUVECs were pretreated with 100 ng/ml PTX for 15 h.

 
In addition, BKCa channel activation by S1P was not inhibited by GDPbetaS and PTX. The pretreatment of HUVECs with PTX did not inhibit BKCa channel activation by S1P (Fig. 8). BKCa channels activities were observed in inside-out patches at 20 mV. The channels were activated by clamping [Ca2+]b at 0.5 µM, and S1P (1 µM) was then applied in the bathing solution. On S1P application, BKCa channel activity was increased (n = 4; Fig. 8, A and C), but current amplitudes were unchanged (Fig. 8B). S1P significantly increased mean open time and open probability from 3.6 ± 0.8 ms and 0.032 ± 0.015 to 7.6 ± 2.2 ms (n = 4; P < 0.05) and 0.139 ± 0.003, respectively (n = 4; P < 0.001). Furthermore, BKCa channel activation by S1P was not inhibited by GDPbetaS (Fig. 9). BKCa channels activities were observed in inside-out patches at 20 mV. The channels were activated by clamping [Ca2+]b at 1 µM, and S1P (1 µM) was then applied in the bathing solution. S1P application increased BKCa channel activity (Fig. 9). When the S1P-induced increase in BKCa channel activity was reached at a steady state, GDPbetaS (1 mM) was applied in the bathing solution. The increased BKCa channel activity by S1P was not affected by GDPbetaS (Fig. 9).


Figure 8
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  		Fig. 8. G protein-independent activation of BKCa channels by S1P in inside-out patches. BKCa currents were activated by loading [Ca2+]i at 0.5 µM, and cells were pretreated with 100 ng/ml PTX for 15 h. A: NPo, which was calculated every 51.2 ms (512-point sweeps), is plotted against time. B and C: current traces (B) recorded at periods labeled a, b, c, and d in A, and their corresponding histograms (C). 01 and 02 indicate the opening level of 1 and 2 channels. NPo, i, and To during periods a–d are given.

 

Figure 9
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  		Fig. 9. G protein-independent activation of BKCa channels by S1P in inside-out patches. BKCa currents were activated by loading [Ca2+]i at 1 µM. A: NPo, which was calculated every 51.2 ms (512-point sweeps), is plotted against time. B and C: current traces (B) recorded at periods labeled a, b, c, and d in A, and their corresponding histograms (C). 01 and 02 indicate the opening level of 1 and 2 channels. NPo, i, and To during periods labeled a–d are given. Note that the activated BKCa channels were not inhibited by GDPbetaS administration.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides evidence to suggest that S1P modulates BKCa channels. The results of the present study indicate that 1) S1P hyperpolarizes the membrane by activating BKCa channels and simultaneously increases [Ca2+]i, and 2) S1P activates BKCa channels independently of GTP and G proteins via a Ca2+-independent sensitization of the channels. From these results, we conclude that S1P activates BKCa channels independently of G protein-coupled receptors. Until now, no evidence had been provided on the modulation of ion channels by S1P independently of G protein-coupled receptors. In addition, our results show that S1P enhances Ca2+ influx by modulating BKCa channels. Inasmuch as S1P is produced in cells by the stimulation of many different receptors, including growth factor receptors and G protein-coupled receptors (30), we suggest that S1P acts a second messenger in Ca2+ signaling in human endothelial cells.

Electrophysiological investigation of Ca2+-activated K+ channel in HUVECs yielded a single-channel conductance of ~200 pS; previous reports indicate a single-channel conductance of 150–250 pS for BKCa channels in endothelial cells (28, 32). According to classification based on single-channel conductance, this channel may therefore be a BKCa channel.

S1P binds to specific cell surface G protein-coupled receptors (13, 21). Thus GTP and G proteins are necessary for S1P action through G protein-coupled receptors (12, 35). In the present study, we found that S1P activates BKCa channels in the absence of GTP and in the presence of the G protein inhibitors, which suggests that S1P activates BKCa channels independently of G protein-coupled receptors. This G protein-independent activation of ion channels by S1P has not been previously reported, whereas sphingosine (the precursor of S1P) has been reported to activate ion channels independently of G protein. Sphingosine activates the melastatin-related cation channel TRPM3 (9) and CRAC (23), whereas these channels are not activated by S1P (9, 23). S1P shifted the Ca2+ concentration-response curve of BKCa channels or BKCa currents leftward, suggesting that BKCa channel or BKCa current activation occurs via an increase in apparent Ca2+ sensitivity.

In endothelial cells, the activations of Ca2+-activated K+ channels, including BKCa channels and intermediate-conductance Ca2+-activated K+ channels, hyperpolarizes the membrane, which increases the driving force for Ca2+ influx (1). Thus membrane potential affects [Ca2+]i by changing the driving force for Ca2+ influx in endothelial cells (20, 33). In the present study, we demonstrated that S1P modulates [Ca2+]i intracellularly as a second messenger by activating BKCa channels independently of S1P receptors. Intracellular S1P either can be secreted or can diffuse across the plasma membrane and activate cell surface S1P receptors in an autocrine or paracrine manner (14). Inasmuch as S1P activates Ca2+ entry channels such as NSC (26) and store-operated Ca2+ entry channels (25) via a PTX-sensitive G protein, S1P might activate Ca2+ entry channels extracellularly via S1P receptors. Thus the extracellular effect of S1P on Ca2+ entry channels might be potentiated by the intracellular effect of S1P on BKCa channels.

The fatty acid-induced modulation of BKCa channels has been studied and found to be lipid-type dependent (2–4, 6). Fatty acids and other negatively charged lipids were found to activate BKCa channels in rabbit pulmonary artery, whereas neutral and short-chain lipids did not affect, and positively charged lipids inhibited, these channels (4). It was suggested that fatty acids and other charged lipids modulate BKCa channel activities by interacting with the channel protein itself. However, mechanisms underlying the activation of BKCa channels by S1P remain to be investigated. Interestingly, sphingosine was found to inhibit a BKCa channel in rabbit pulmonary arterial cells in inside-out patches (4), which contradicts the results of the present study, in which sphingosine was found to activate a BKCa channel in HUVECs. This discrepancy may have been caused by structural differences between BKCa channels in endothelium and vascular smooth muscle cells. BKCa channels in endothelial cells are composed of {alpha}-subunits not associated with beta-subunits (31). This lack of beta-subunit involvement indicates a substantially different form of channel regulation in endothelial cells versus vascular smooth muscle cells (31).

S1P is a blood constituent released from activated platelets (16) and is present in human plasma and serum at concentrations of 200 and 500 nM, respectively, and thus human endothelial cells are constantly exposed to S1P (40). The EC50 of S1P in terms of BKCa current activation is ~400 nM, and the concentration of S1P in plasma or serum is close to this value. However, the active concentration of S1P might be less than its concentration, because >60% of S1P is localized to the lipoprotein fraction (27) and interactions between S1P and lipoproteins reduce its active concentration.

Because of the polar nature of the S1P head group, it cannot transverse the cell membrane readily (11). In previous studies, it was proposed that S1P directly releases Ca2+ from intracellular stores by acting as an intracellular second messenger (24, 36). One of the possible explanations for this is that G protein-coupled receptors on the cell surface are activated by S1P in the external solution (14) and that S1P is subsequently produced in cells by these activated receptors (30). However, this explanation is not plausible, because BKCa channels were activated independently of G protein-coupled receptors in the present study. On the other hand, if it is assumed that specific transporters for S1P exist, then S1P can readily transverse the plasma membrane (14).

In conclusion, the present study shows that S1P activates BKCa channels independently of G protein-coupled receptors and therefore modulates Ca2+ driving force. Inasmuch as S1P activates various Ca2+ entry channels such as CRAC, store-operated Ca2+ channels, and NSC, Ca2+ entry through these channels might be fine-tuned by regulating the membrane potential.


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This work was supported by Grant R01-2003-000-10466-0 from the Basic Research Program of the Korea Science & Engineering Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. H. Suh, Dept. of Physiology, College of Medicine, Ewha Woman's Univ., 911-1 Mok-6-dong, Yang Chun-gu, Seoul, Republic of Korea, 158-710 (e-mail: shsuh{at}ewha.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 REFERENCES
 
1. Ahn SC, Seol GH, Kim JA, and Suh SH. Characteristics and a functional implication of Ca2+-activated K+ current in mouse aortic endothelial cells. Pflügers Arch 447: 426–435, 2004.[CrossRef][Web of Science][Medline]

2. Baron A, Frieden M, and Beny JL. Epoxyeicosatrienoic acids activate a high-conductance, Ca2+-dependent K+ channel on pig coronary artery endothelial cells. J Physiol 504: 537–543, 1997.[Abstract/Free Full Text]

3. Chang HM, Reitstetter R, and Gruener R. Lipid-ion channel interactions: increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior. J Membr Biol 145: 13–19, 1995.[Web of Science][Medline]

4. Clarke AL, Petrou S, Walsh JV Jr, and Singer JJ. Modulation of BKCa channel activity by fatty acids: structural requirements and mechanism of action. Am J Physiol Cell Physiol 283: C1441–C1453, 2002.[Abstract/Free Full Text]

5. Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, and Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381: 800–803, 1996.[CrossRef][Medline]

6. Denson DD, Wang X, Worrell RT, and Eaton DC. Effects of fatty acids on BK channels in GH3 cells. Am J Physiol Cell Physiol 279: C1211–C1219, 2000.[Abstract/Free Full Text]

7. Goodemote KA, Mattie ME, Berger A, and Spiegel S. Involvement of a pertussis toxin-sensitive G protein in the mitogenic signaling pathways of sphingosine-1-phosphate. J Biol Chem 270: 10272–10277, 1995.[Abstract/Free Full Text]

8. Gratzinger D, Canosa S, Engelhardt B, and Madri JA. Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho. FASEB J 17: 1458–1469, 2003.[Abstract/Free Full Text]

9. Grimm C, Kraft R, Schultz G, and Harteneck C. Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine. Mol Pharmacol 67: 798–805, 2005.[Abstract/Free Full Text]

10. Hla T. Physiological and pathological actions of sphingosine-1-phosphate. Semin Cell Dev Biol 15: 513–520, 2004.[CrossRef][Web of Science][Medline]

11. Hla T. Signaling and biological actions of sphingosine-1-phosphate. Pharmacol Res 47: 401–407, 2003.[CrossRef][Web of Science][Medline]

12. Hla T. Sphingosine-1-phosphate receptors. Prostaglandins 64: 135–142, 2001.[Web of Science][Medline]

13. Hla T, Lee MJ, Ancellin N, Paik JH, and Kluk MJ. Lysophospholipids—receptor revelations. Science 294: 1875–1878, 2001.[Abstract/Free Full Text]

14. Hobson JP, Rosenfeldt HM, Barak LS, Olivera A, Poulton S, Caron MG, Milstien S, and Spiegel S. Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility. Science 291: 1800–1803, 2001.[Abstract/Free Full Text]

15. Igarashi J and Michel T. Sphingosine-1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways. J Biol Chem 276: 36281–36288, 2001.[Abstract/Free Full Text]

16. Igarashi Y and Yatomi Y. Sphingosine-1-phosphate is a blood constituent released from activated platelets, possibly playing a variety of physiological and pathophysiological roles. Acta Biochim Pol 45: 299–309, 1998.[Web of Science][Medline]

17. Ishii I, Fukushima N, Ye X, and Chun J. Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 73: 321–354, 2004.[CrossRef][Web of Science][Medline]

18. Itagaki K and Hauser CJ. Sphingosine-1-phosphate, a diffusible Ca2+ influx factor mediating store-operated Ca2+ entry. J Biol Chem 278: 27540–27547, 2003.[Abstract/Free Full Text]

19. Jaffe EA, Nachman RL, Becker CG, and Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52: 2745–2756, 1973.[Web of Science][Medline]

20. Kamouchi M, Droogmans G, and Nilius B. Membrane potential as a modulator of the free intracellular Ca2+ concentration in agonist-activated endothelial cells. Gen Physiol Biophys 18: 199–208, 1999.[Web of Science][Medline]

21. Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, and Hla T. Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science 279: 1552–1555, 1998.[Abstract/Free Full Text]

22. Levade T, Auge N, Veldman RJ, Cuvillier O, Negre-Salvayre A, and Salvayre R. Sphingolipid mediators in cardiovascular cell biology and pathology. Circ Res 89: 957–968, 2001.[Abstract/Free Full Text]

23. Mathes C, Fleig A, and Penner R. Calcium release-activated calcium current (ICRAC) is a direct target for sphingosine. J Biol Chem 273: 25020–25030, 1998.[Abstract/Free Full Text]

24. Mattie M, Brooker G, and Spiegel S. Sphingosine-1-phosphate, a putative second messenger, mobilizes Ca2+ from internal stores via an inositol trisphosphate-independent pathway. J Biol Chem 269: 3181–3188, 1994.[Abstract/Free Full Text]

25. Mehta D, Konstantoulaki M, Ahmmed GU, and Malik AB. Sphingosine-1-phosphate-induced mobilization of intracellular Ca2+ mediates rac activation and adherens junction assembly in endothelial cells. J Biol Chem 280: 17320–17328, 2005.[Abstract/Free Full Text]

26. Muraki K and Imaizumi Y. A novel function of sphingosine-1-phosphate to activate a non-selective cation channel in human endothelial cells. J Physiol 537: 431–441, 2001.[Abstract/Free Full Text]

27. Murata N, Sato K, Kon J, Tomura H, Yanagita M, Kuwabara A, Ui M, and Okajima F. Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochem J 352: 809–815, 2000.[CrossRef][Web of Science][Medline]

28. Nilius B and Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415–1459, 2001.[Abstract/Free Full Text]

29. Nilius B, Schwarz G, Oike M, and Droogmans G. Histamine-activated, non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein. Pflügers Arch 424: 285–293, 1993.[CrossRef][Web of Science][Medline]

30. Olivera A and Spiegel S. Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins 64: 123–134, 2001.[Web of Science][Medline]

31. Papassotiriou J, Kohler R, Prenen J, Krause H, Akbar M, Eggermont J, Paul M, Distler A, Nilius B, and Hoyer J. Endothelial K+ channel lacks the Ca2+ sensitivity-regulating beta subunit. FASEB J 14: 885–894, 2000.[Abstract/Free Full Text]

32. Rusko J, Tanzi F, van Breemen C, and Adams DJ. Ca2+-activated K+ channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block. J Physiol 455: 601–621, 1992.[Abstract/Free Full Text]

33. Seol GH, Ahn SC, Kim JA, Nilius B, and Suh SH. Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility. Am J Physiol Heart Circ Physiol 286: H329–H339, 2004.[Abstract/Free Full Text]

34. Spiegel S and Milstien S. Sphingosine-1-phosphate: signaling inside and out. FEBS Lett 476: 55–57, 2000.[CrossRef][Web of Science][Medline]

35. Taha TA, Argraves KM, and Obeid LM. Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy. Biochim Biophys Acta 1682: 48–55, 2004.[Medline]

36. Van Brocklyn JR, Lee MJ, Menzeleev R, Olivera A, Edsall L, Cuvillier O, Thomas DM, Coopman PJ, Thangada S, Liu CH, Hla T, and Spiegel S. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 142: 229–240, 1998.[Abstract/Free Full Text]

37. Wang J, Carbone LD, and Watsky MA. Receptor-mediated activation of a Cl– current by LPA and S1P in cultured corneal keratocytes. Invest Ophthalmol Vis Sci 43: 3202–3208, 2002.[Abstract/Free Full Text]

38. Wang Y, Adair CD, Coe L, Weeks JW, Lewis DF, and Alexander JS. Activation of endothelial cells in preeclampsia: increased neutrophil-endothelial adhesion correlates with up-regulation of adhesion molecule P-selectin in human umbilical vein endothelial cells isolated from preeclampsia. J Soc Gynecol Investig 5: 237–243, 1998.[CrossRef][Web of Science][Medline]

39. Yamaguchi H, Kitayama J, Takuwa N, Arikawa K, Inoki I, Takehara K, Nagawa H, and Takuwa Y. Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells. Biochem J 374: 715–722, 2003.[CrossRef][Web of Science][Medline]

40. Yatomi Y, Igarashi Y, Yang L, Hisano N, Qi R, Asazuma N, Satoh K, Ozaki Y, and Kume S. Sphingosine-1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum. J Biochem (Tokyo) 121: 969–973, 1997.[Abstract/Free Full Text]





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