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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Sphingosine 1-Phosphate Causes Airway Hyper-Reactivity by Rho-Mediated Myosin Phosphatase Inactivation

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

Received July 18, 2006; accepted November 10, 2006.

	Abstract

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In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F₃₄₀/F₃₈₀). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F₃₄₀/F₃₈₀. This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca²⁺ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca²⁺ sensitization via inactivation of myosin phosphatase, which links G_i and RhoA/Rho-kinase processes.

External application of S1P to smooth muscle in a various tissues causes contraction with an increase in the concentrations of intracellular Ca²⁺ ([Ca²⁺]_i) (Watterson et al., 2005). The Ca²⁺ mobilization induced by S1P is mediated by Ca²⁺ influx pass through voltage-dependent L-type Ca²⁺ channels and store-operated Ca²⁺ entry (Ghosh et al., 1990; Bischoff et al., 2000,Bischoff et al., 2000; Rosenfeldt et al., 2003). On the other hand, Rho, a small GTPase, and Rho-kinase, a specific effecter of Rho, are involved in the postreceptor signal transduction pathways of S1P (Coussiun et al., 2002; Rosenfeldt et al., 2003; Salomone et al., 2003; Zhou and Murthy, 2004). However, in smooth muscle contraction induced by S1P, the role of Ca²⁺ sensitization that relates to Rho/Rho-kinase pathways remains unclear. Extracellular S1P acts as a ligand for a family of specific G-protein coupled receptors (S1P_1–5), which are coupled to a variety of G proteins that are linked, in turn, to downstream signaling pathways (Payne et al., 2002; Siehler and Manning, 2002). In the respiratory system, including airway smooth muscle (ASM), it is generally considered that S1P₂ may play an important physiological role (Rosenfeldt et al., 2003). Previous studies in cultured human ASM cells using mechanically loaded collagen matrix have shown that S1P causes contraction by activating voltage-dependent L-type Ca²⁺ channels (Rosenfeldt et al., 2003). Moreover, the S1P-induced contraction in ASM cells is mediated by pertussis toxin (PTX)-insensitive processes (Rosenfeldt et al., 2003). However, it has been proposed that G_i may participate in S1P-induced contraction of smooth muscle in other tissues (Bischoff et al., 2000,Bischoff et al., 2000; Salomone et al., 2003; Zhou and Murthy, 2004). Little is currently known about S1P action in ASM, although it could act as a mediator of the interaction between inflammatory cells and ASM in bronchial asthma.

This study was designed to determine whether extracellular S1P is involved in airway hyper-reactivity, which is implicated in the pathophysiology of bronchial asthma. To identify the molecular mechanisms of regulating ASM contractility following continuous exposure to S1P, we focused on the role of Ca²⁺ mobilization and Ca²⁺ sensitization in the postreceptor signal transduction pathways activated by S1P.

	Materials and Methods

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Tissue Preparation and Solution. Male Hartley guinea pigs (300–350 g) were killed by injection of overdose of anesthetics (150 mg/kg pentobarbital i.p.), and tracheas were excised. The tracheal rings were opened by cutting longitudinally at the cartilaginous region, and the epithelium was dissected out. The normal bathing solution was composed of 137 mM NaCl, 5.9 mM KHCO₃, 2.4 mM CaCl₂, 1.2 mM MgCl₂, and 11.8 mM glucose, bubbled with a mixture of 99% O₂ and 1% CO₂ (pH 7.4). The organ bath was filled with the bathing solution at a constant flow of 3 ml/min, and the temperature of the organ bath was maintained at 37°C. All animal procedures were approved by The Animal Care and Use Committee, Nagoya University Graduate School of Medicine.

Isometric Tension Recording and Measurement of Fura-2 Fluorescence. Isometric tension and fura-2 fluorescence were measured essentially as described previously (Kume and Takagi, 1997; Kume et al., 2001). Muscle strips containing four cartilaginous rings, one for isometric tension recording and three for measurement of [Ca²⁺]_i, were prepared. Muscle strips were treated with 10 µM acetoxymethyl ester of fura-2 for 4 h at room temperature (22–24°C). The noncytotoxic detergent, Pluronic F-127 (0.01% w/v), was added to increase the solubility of fura-2. After the loading, the chamber was filled with the normal bating solution at 37°C for 50 min to wash out the extracellular fura-2 prior to the measurements. Isometric tension and the fura-2 fluorescence of the muscle strips were measured simultaneously, using a displacement transducer and a spectrofluorometer (CAF-110; Japan Spectroscopic, Tokyo, Japan). The intensities of fluorescence due to excitation at 340 nm (F₃₄₀) and 380 nm (F₃₈₀) were measured after background subtraction. The absolute amount of [Ca²⁺]_i was not calculated because the dissociation constant of fura-2 for Ca²⁺ in smooth muscle cytoplasm is different from that obtained in vitro (Konishi et al., 1988). Therefore, the ratio F₃₄₀/F₃₈₀ was used as a relative indicator of [Ca²⁺]_i. Muscle tension and F₃₄₀/F₃₈₀ in the resting state were taken as 0%, and the values of percent contraction and percent F₃₄₀/F₃₈₀ were expressed by taking response to 1 µM methacholine (MCh) or 10 µM histamine at each experimental condition as 100%. Time-matched control tissues were treated similarly to the test tissues but exposed continuously to the normal bathing solution instead of S1P and PTX. The resting tone was abolished by addition to 2 µM indomethacin throughout the experiments.

Cell Culture. Primary cultures of normal human bronchial smooth muscle (BSM) cells from multiple donors were obtained from Cambrex (Walkersville, MD). The cells were maintained in culture medium containing 5% fetal bovine serum, human recombinant epidermal growth factor (1 ng/ml), insulin (10 mg/ml), human recombinant fibroblast growth factor (2 ng/ml), gentamicin (50 mg/ml), and amphotericin B (0.05 mg/ml) (SmGM-2 BulletKit; Cambrex) in an atmosphere of 5% CO₂ and 95% air at 37°C. Cells at the 5th to 7th passages were used for the experiments.

RhoA Activation Assay. The amount of activated Rho (GTP-Rho) was determined using a Rho activation assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). The human BSM cells were grown to confluence and then placed in serum-free medium (Dulbecco's modified Eagle's medium/F-12; Invitrogen, Carlsbad, CA) containing antibiotics-antimycotic (100 units/ml penicillin, 100 µl/ml streptomycin, 250 ng/ml amphotericin B; Invitrogen) for 24 h. After stimulation by S1P, the cells were washed with ice-cold phosphate-buffered saline (PBS). Cellular lysates were prepared according to the manufacturer's instructions.

Phosphorylation of Myosin-Binding Subunit. Phosphorylation of myosin-binding subunit (MBS) was evaluated with specific antibodies for myosin phosphatase target subunit 1 (MYPT1). The human BSM cells were grown to confluence and then placed in serum-free medium for 24 h. After exposure to agents, the BSM cells were washed with ice-cold PBS. Whole-cell lysates were prepared by treating the cells with lysis buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, and 5% 2-mercaptoethanol).

Western Blot. Protein contents of cellular lysates were measured using a Bio-Rad protein assay reagent kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Equal amounts of lysates, adjusted to protein content, were resolved by SDS-polyacrylamide gel electrophoresis using a 4 to 20% linear gradient running gel. Proteins were transferred to a nitrocellulose membrane, and the membrane was incubated at room temperature in PBS containing 0.2% Tween 20 for 1 h. Immunoblotting was performed using antibodies against RhoA, MYPT1 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-phospho-MYPT1 (Thr⁸⁵⁰) (Upstate Biotechnology, Inc.). Immunodetection was accomplished using a sheep anti-mouse secondary antibody or donkey anti-rabbit secondary antibody and the enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). The intensity was quantified using Scion image software (Scion Image, Frederick, MD).

Experimental Protocols. To examine the effects of S1P on ASM, the fura-2-loaded tissues of guinea pig tracheal smooth muscle were treated with S1P for 15 min. Before and after application of S1P, the strips were washed out for 10 min with the normal bathing solution. To examine the effects of S1P on airway hyper-reactivity, the contraction and F₃₄₀/F₃₈₀ in response to 1 µM MCh and 10 µM histamine were measured before and after exposure to S1P. To determine the involvement of Ca²⁺ mobilization by S1P in the pathogenesis of airway hyper-reactivity, the tension and F₃₄₀/F₃₈₀ in response to MCh were examined before and after exposure to S1P in the absence and presence of Ca²⁺ channel blockers such as SKF96365, a nonselective inhibitor of Ca²⁺ channels, and verapamil, a selective inhibitor of voltage-dependent Ca²⁺ channels. To determine the involvement of Ca²⁺ sensitization by S1P in these mechanisms, response to MCh was similarly examined in the absence and presence of Y-27632, a selective inhibitor of Rho-kinase, PD98059, an inhibitor of mitogen-activated protein kinase kinase, and bisindolylmaleimide, an inhibitor of protein kinase C. When the period for incubation with S1P (<100 nM) was lengthened to 6 h, the strips were not loaded with fura-2. To determine the involvement of G_i, the inhibitory G protein of adenylyl cyclase, in the augmentation of MCh-induced contraction by S1P, the strips were pretreated with 1 µg/ml PTX for 6 h, and then the response to MCh was examined after exposure to S1P. To determine relationships between S1P and RhoA, after treating the human BSM cells with 3 µM S1P for 15 min, GTP-RhoA was measured using the RhoA activation assay. Moreover, to confirm the effects of S1P on RhoA, phosphorylation of MYPT1, a direct target substrate of Rho-kinase, was measured in the human BSM cells using Western blots. To determine the involvement of Rho-kinase and extracellular signal-regulated kinase in the stimulation of MYPT1 phosphorylation by S1P, the cells were treated with Y-27632 and PD98059, respectively, 30 min before stimulation by S1P. To determine the involvement of G_i in this phosphorylation, the cells were treated with 100 ng/ml PTX for 4 h before stimulation with S1P.

Agents. MCh, indomethacin, PTX, verapamil, bisindolylmaleimide, histamine, and Pluronic F-127 were obtained from Sigma (St. Louis, MO). S1P was obtained from BIOMOL (Plymouth Meeting, PA). Y-27632 was obtained from Wako (Osaka, Japan). SKF96365 and PD98059 were obtained from Calbiochem (La Jolla, CA). Fura-2/AM was obtained from Dojin Laboratories (Kumamoto, Japan).

Statistics. All data were expressed as means ± S.D., and response to an agent under each experimental condition was described as the percentage of the control condition. Values of concentration of agents that produces a 50% response (EC₅₀) of contraction by 1 µM MCh were determined using regression analysis applied to the linear portion of each concentration-response curve. Statistical significance was assessed by Student's t test or repeated-measures analysis of variance, followed by Bonferroni post hoc test. p < 0.05 was considered to be a significant difference.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Augmentation of MCh-induced contraction by S1P in ASM. A, typical example of a simultaneous record of tension (top trace) and F340/F380 (bottom trace) in response to 1 µM MCh before and after 3 µM S1P for 15 min. B, concentration-response curves for S1P on tension (open circles) and F340/F380 (closed circles). The values of percent response to S1P were expressed taking response to 1 µM MCh as 100%. C, values of percent contraction (open columns) and F340/F380 (closed columns) for MCh after exposure to the normal bathing solution (control) and S1P between 0.03 and 3 µM for 15 min. D, values of percent contraction (open columns) and F340/F380 (closed columns) for histamine after exposure to S1P in the same way in C. E, concentration-response curves for MCh after exposure to the normal bathing solution (open circles), 0.003 µM (closed circles), and 0.03 µM S1P (open squares) for 6 h. The abscissas in B and E express molar concentrations on a log scale. *, p < 0.05; **, p < 0.01

	Results

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View larger version (22K): [in this window] [in a new window]   Fig. 2. Involvement of the Rho/Rho-kinase pathway in the hyperresponsiveness to MCh induced by S1P. A, typical example of a simultaneous record of tension (top trace) and F340/F380 (bottom trace) in response to 1 µM MCh before and after 3 µM S1P for 15 min in the presence of 1 µM Y-27632. B, values of percent contraction (open columns) and F340/F380 (closed columns) for MCh after exposure to the normal bathing solution (control) and S1P in the presence of Y-27632 between 0.01 and 1.0 µM. C, concentration-response curves for MCh after exposure to 0.03 µM S1P in the absence (open circles) and presence of Y-27632 (0.1 µM; closed circles) and 1.0 µM (open squares) for 6 h. D, those values of percent contraction (open columns) and F340/F380 (closed columns) for MCh after exposure to S1P in the absence (control) and presence of 30 µM PD98059 and 10 µM bisindoylmaleimide (BIS) for an equivalent minute. The abscissa in C expresses molar concentrations on a log scale. *, p < 0.05; **, p < 0.01.

Next, in the presence of 30 µM PD98059, response to MCh was examined before and after exposure to 3 µM S1P for 15 min. However, under this experimental condition, the contraction induced by 1 µM MCh following exposure to S1P was increased without an elevation in F₃₄₀/F₃₈₀, similar to the result shown in Fig. 1A. The values of percent contraction and percent F₃₄₀/F₃₈₀ for MCh were 134.9 ± 12.4 (not significant) and 105.2 ± 9.2% (not significant), respectively (n = 6, Fig. 2D). In the presence of 10 µM bisindoylmaleimide, MCh-induced contraction after exposure to S1P was also augmented without a change in F₃₄₀/F₃₈₀. The values of percent contraction and percent F₃₄₀/F₃₈₀ for MCh were 131.2 ± 14.4 (not significant) and 102.2 ± 9.7% (not significant), respectively (n = 6, Fig. 2D).

Ca²⁺ Mobilization Is Not Involved in the Induction of Airway Hyper-Reactivity by S1P. SKF96365 (100 µM) resulted in a marked suppression of both tension and F₃₄₀/F₃₈₀ induced by 3 µM S1P (Fig. 3A). However, after exposure to S1P with SFK96365, 1 µM MCh-induced contraction was markedly increased without elevating F₃₄₀/F₃₈₀, similar to the result shown in Fig. 1A. The values of percent contraction and percent F₃₄₀/F₃₈₀ for MCh after exposure to S1P with SKF96365 were 129.7 ± 12.9% (n = 6, not significant) and 96.3 ± 9.8% (n = 6, not significant), respectively (Fig. 3B). Verapamil (10 µM) caused a modest inhibition in the augmented tension and F₃₄₀/F₃₈₀ by equimolar S1P (data not shown). Moreover, after exposure to S1P in the presence of verapamil, 1 µM MCh-induced contraction was markedly increased independent of F₃₄₀/F₃₈₀, similar to the results shown in Fig. 1A. The values of percent contraction and percent F₃₄₀/F₃₈₀ for MCh after exposure to S1P in the presence of verapamil were 134.1 ± 12.1 (n = 6, not significant) and 102.4 ± 13.1% (n = 6, not significant), respectively (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   Fig. 3. S1P-induced airway hyper-reactivity is not involved in Ca2+ channel activity. A, typical example of a simultaneous record of tension (top trace) and F340/F380 (bottom trace) in response to 1 µM MCh before and after 3 µM S1P for 15 min in the presence of 100 µM SKF96365. B, values of percent contraction (open columns) and F340/F380 (closed columns) for MCh after exposure to S1P in the absence (control) and presence of an equimolar of SKF96365 and 3 µM verapamil for an equivalent time.

Involvement of G_i in the Induction of Airway Hyper-Reactivity Induced by S1P. Pretreatment of the fura-2-unloaded tissues with PTX 1 µg/ml for 6 h caused a marked attenuation of the augmented response to 1 µM MCh after exposure to 3 µM S1P in a time-dependent manner (Figs. 4, A and B). The values of percent contraction for MCh after exposure to S1P, followed by treatment without and with PTX for 6 h, were 134.6 ± 12.8 (n = 8) and 102.5 ± 8.6% (n = 8, p < 0.01), respectively (Fig. 4B). MCh (0.001–10 µM) was cumulatively applied to the tissues before and after incubation with 0.1 and 1.0 µg/ml PTX in the presence of 0.03 µM S1P for 6 h. The concentration-response curves for MCh after exposure to S1P with PTX were shifted to the right in a concentration-dependent manner (Fig. 4C). The values of EC₅₀ for the curves for MCh after exposure to S1P without and with 1.0 µg/ml PTX for 6 h were 0.08 ± 0.05 (n = 8) and 0.48 ± 0.18 µM (n = 8, p < 0.01), respectively (Fig. 4C).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Involvement of Gi in the induction of airway hyper-reactivity induced by S1P. A, typical record of tension by 1 µM MCh before and after exposure to 3 µM S1P for 15 min subsequent to incubation with 1.0 µg/ml PTX (top trace) and the normal bathing solution (sham, bottom trace) for 6 h. B, values of percent contraction for MCh after exposure to 3 µM S1P subsequent to incubation with the normal bathing solution (control), and an equivalent concentration of PTX for 2 to 6 h. C, concentration-response curves for MCh after exposure to 0.03 µM S1P in the absence (open circles) and presence of 0.1 µg/ml (closed circles), and 1.0 µg/ml (open squares) PTX for 6 h. The abscissa in C expresses molar concentrations on a log scale. *, p < 0.05; **, p < 0.01.

View larger version (40K): [in this window] [in a new window]   Fig. 5. The effects of S1P on RhoA activity in the human BSM cells. Human BSM cells were incubated with the medium in the absence (control) and presence of 1 µM S1P for 15 min. Activated form of Rho (GTP-Rho) was pulled down from the cell lysates with Rhotekin-binding domain-conjugated beads and analyzed by Western blotting using anti-RhoA. The identical cell lysates were used to determine total Rho. Data are representative of four independent experiments (top trace). The value of RhoA-GTP/total RhoA by S1P is calculated relative to that for control 1 (bottom trace). **, p < 0.01.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Regulation of MBS phosphorylation by S1P. Phosphorylated MBS in human BSM cells was evaluated with immunoblotting using anti-MYPT1 (Thr850). A, effects of S1P on MBS phosphorylation. B, inhibitory effects of Y-27632 on phosphorylated MBS by S1P. C, effects of PTX and PD98059 on phosphorylated MBS by S1P. The values of p-MYPT1/MYPT1 are calculated relative to that for control at each experimental condition (open columns). *, p < 0.05; **, p < 0.01 (versus response to control); , p < 0.01 (versus response to S1P).

	Discussion

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The contractile effect of S1P on ASM has not been previously investigated in tissue samples, although studies in vascular smooth muscle have demonstrated that S1P induces contraction at concentrations above 100 nM (Bischoff et al., 2000,Bischoff et al., 2000; Tosaka et al., 2001; Salomone et al., 2003), similar results were observed in ASM (Fig. 1B). Although the level of S1P in BAL fluid is elevated by an antigen challenge in patients with asthma, the concentration is less than 100 nM (Ammit et al., 2001; Jolly et al., 2001). Hence, extracellular S1P may not have a direct role in bronchoconstriction associated with bronchial asthma. Pretreatment with concentrations of S1P below 100 nM for 15 min also did not enhance subsequent responses to MCh or histamine (Fig. 1, A, C, and D). However, when the period of incubation with S1P was extended to 6 h, lower concentrations of S1P (3–30 nM), which are similar to the level attained in BAL fluid after an antigen challenge, caused a marked enhancement of the response to MCh (Fig. 1E). These results in isolated guinea pig ASM tissues were also observed in isolated human ASM tissues effects (data not shown). Therefore, although whether S1P is related to airway hyper-reactivity in patients with asthma remains unknown, our results suggest that extracellular S1P, which is released from inflammatory cells, can lead to airway hyper-reactivity clinically.

As shown in Figs. 1B and 3A, S1P is less potent in enhancing the sensitivity to intracellular Ca²⁺ than MCh, and Ca²⁺ influx passing through Ca²⁺ channels nonselectively plays a functional important role in S1P-induced contraction. S1P has modest effects on verapamil-sensitive Ca²⁺ channels. In addition, similar to our observations, previous patch-clamp studies have shown that in endothelial cells, S1P activates nonselective cation channels (Muraki and Imaizumi, 2001). In contrast, our results indicate that Ca²⁺ sensitization mediated by Rho-kinase is involved in the functional effects of S1P on ASM (Fig. 2A). External application of S1P augments RhoA activity and the phosphorylation of MYPT1 (Figs. 5 and 6), demonstrating that a loss of MP activity via activation of the RhoA/Rho-kinase processes plays an important role in the action of S1P in ASM. Although a few reports have shown that S1P phosphorylates myosin light chain in smooth muscle-containing airways (Rosenfeldt et al., 2003; Zhou and Murthy, 2004), the main action of Rho-kinase in Ca²⁺ sensitization appears to be the inhibition in MP activity due to phosphorylation of MBS (Kimura et al., 1996; Matsui et al., 1996; Fukata et al., 2001). As shown in this study, Rhokinase acts on MYPT1 in ASM, which is similar to findings in vascular smooth muscle (Velasco et al., 2002; Liu et al., 2004; Wilson et al., 2005). Therefore, phosphorylation of myosin light chain is an indirect response to S1P as a result of inhibition in MP activity by Rho-kinase.

We also examined the involvement of Ca²⁺ mobilization and Ca²⁺ sensitization in the induction of airway hyper-reactivity after exposure to S1P. As shown in Fig. 1, A and C, Ca²⁺ sensitization is involved in an increase in MCh-induced contraction after exposure to S1P. When S1P-induced contraction was suppressed in the presence of Y-27632 independent of [Ca²⁺]_i, the augmented response to MCh after exposure to S1P was abolished in a concentration-dependent manner (Fig. 2, A and B). Moreover, Y-27632 also suppressed the ability of an extended treatment with the clinical levels of S1P to enhance the effects of MCh (Fig. 2C). Therefore, Ca²⁺ sensitization via Rho-kinase-dependent inactivation of MP participates in the ability of S1P to induce airway hyper-reactivity. In contrast, even though both tension and [Ca²⁺]_i by S1P was suppressed in the presence of SKF96365 and verapamil, the ability of S1P to enhance MCh-induced contraction was unaffected by these Ca²⁺ antagonists (Fig. 3, A and B). These results provide further support of the idea that Ca²⁺ sensitization but not Ca²⁺ mobilization is involved in the effect of S1P on the induction of airway hyper-reactivity in ASM. However, as shown in Fig. 2D, extracellular signal-regulated kinase and protein kinase C do not appear to be involved in the Ca²⁺ sensitization by S1P (Shirao et al., 2002).

Next, we sought to determine the involvement of the receptor and postreceptor signal transduction pathways in the induction of airway hyper-reactivity after treatment with S1P. As shown in Fig. 4, A to C, preincubation with PTX causes an inhibition in the enhancement of MCh-induced contraction by S1P in time- and concentration-dependent manners. Since exposure to PTX causes a functional uncoupling of G_i, the inhibitory heterotrimeric G protein of adenylyl cyclase, from its associated receptor via the ADP ribosylation, G_i may mediate the ability of S1P to cause hyper-reactivity to MCh. A previous report has indicated that MCh-induced contraction in ASM is partly attenuated after exposure to PTX (Kume et al., 1995). Although the inhibitory effects of PTX on the S1P-induced hyper-reactivity to MCh may be due to muscarinic inhibition by PTX, incubation with PTX directly suppressed the phosphorylation of MYPT1 by S1P (Fig. 6C). Moreover, in ASM, the hyperresponsiveness to histamine was similarly observed by pretreatment with S1P (Fig. 1D). These results indicate that an inhibition in the muscarinic hyper-reactivity by S1P is due to a PTX-sensitive pathway that alters the action of S1P rather than a direct effect of the PTX-sensitive pathway on muscarinic action. In endothelial cells, activation of nonselective cation channels by S1P is also suppressed by pretreatment with PTX (Muraki and Imaizumi, 2001). Although it remains unclear whether G_i is involved in the effect of S1P on ASM, our results demonstrate that G_i is related to S1P-induced hyper-reactivity to MCh via activation of MP. In smooth muscle other than ASM, external application of S1P augmented both force generation and [Ca²⁺]_i via a PTX-sensitive process (Bischoff et al., 2000,Bischoff et al., 2000; Salomone et al., 2003; Zhou and Murthy, 2004). Moreover, in nonsmooth muscle, the effect of S1P is antagonized by PTX (Liu et al., 2001; Rouach et al., 2006). As shown in Fig. 6, the phosphorylation of MYPT1 was markedly attenuated after exposure to 0.1 µg/ml PTX for 4 h. In ASM cells, the inhibitory effects of MCh on Ca²⁺-activated K⁺ channel activity is eliminated by a 4-h pretreatment with PTX (Kume and Kotlikoff, 1991; Kume et al., 1992). In ASM tissues, in contrast, more than 4 h are needed for the maximal effect of PTX.

It is generally considered that S1P₃ receptors are coupled to G_i (Ancellin and Hla, 1999; Hla et al., 2001; Siehler and Manning, 2002) and that it enhances Rho activity (Paik et al., 2001) and Ca²⁺ influx (Ishii et al., 2002). Suramin is a nonselective antagonist of S1P₃; however, this agent is also used as an inhibitor of S1P (Ancellin and Hla, 1999). In the presence of suramin, the enhancement of MCh-induced contraction by pretreatment with S1P was markedly attenuated in a concentration-dependent manner (data not shown), suggesting that S1P₃ is involved in the S1P-induced hyperresponsiveness to MCh in ASM. Although the S1P receptor subtype and the postreceptor pathway have not been fully elucidated yet in this study, loss of MP activity by the relationship between S1P₃/G_i and Rho/Rho-kinase may play a fundamentally functional role in the induction of hyper-reactivity to MCh by S1P in ASM.

In conclusion, pretreatment of ASM with the clinical level of S1P leads to an enhancement of contractility of ASM due to Ca²⁺ sensitization via Rho-mediated MP inactivation. Although clinical relevance of these results remains unknown, our observation may provide the evidence that S1P acts as a lipid mediator and that extracellular S1P causes airway hyper-reactivity implicated in the pathophysiology of bronchial asthma.

	Acknowledgements

 
We thank Professor Kozo Kaibuchi (Department of Cell Pharmacology, Nagoya University Graduate School of Medicine) for helpful comments.

	Footnotes

 
This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan (Grants-in-Aid 17590785 to H.K. and 17790531 to S.I.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110718.

ABBREVIATIONS: S1P, sphingosine 1-phosphate; BAL, bronchoalveolar lavage; ASM, airway smooth muscle; PTX, pertussis toxin; MCh, methacholine; BSM, bronchial smooth muscle; PBS, phosphate-buffered saline; MBS, myosin-binding subunit; MYPT1, myosin phosphatase target subunit 1; SKF96365, 1-{-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide; PD98059, 2'-amino-3'-methoxyflavone; MP, myosin phosphatase.

Address correspondence to: Dr. Hiroaki Kume, Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsrumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: hkume{at}med.nagoya-u.ac.jp

	References

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