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Am J Physiol Gastrointest Liver Physiol 291: G605-G610, 2006. First published May 4, 2006; doi:10.1152/ajpgi.00147.2006
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LIVER AND BILIARY TRACT

Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection

Wenhui Hu, Sunila Mahavadi, Jiean Huang, Fang Li, and Karnam S. Murthy

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Viginia

Submitted 30 March 2006 ; accepted in final form 1 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingosine-1-phosphate (S1P) induces an initial Ca2+-dependent contraction followed by a sustained Ca2+-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P1 and S1P2 receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P1 or S1P2 receptors. Phospholipase C (PLC)-beta activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P1 receptors abolished S1P-stimulated activation of G{alpha}i3 and partially inhibited activation of G{alpha}i1, whereas silencing of S1P2 receptors abolished activation of G{alpha}q, G{alpha}13, and G{alpha}i2 and partially inhibited activation of G{alpha}i1. Silencing of S1P2 but not S1P1 receptors suppressed S1P-stimulated PLC-beta and Rho kinase activities, implying that both signaling pathways were mediated by S1P2 receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P1 receptor agonist SEW2871 did not stimulate PLC-beta or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P1 receptors so as to enable chemical inactivation of S1P2 receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P2 receptors. Thus S1P1 and S1P2 receptors are coupled to distinct complements of G proteins. Only S1P2 receptors activate PLC-beta and Rho kinase and mediate initial and sustained contraction.

small interfering RNAs; lentiviral vector; sphingosine-1-phosphate; phospholipase C-beta; Rho kinase

THE LYSOPHOSPHOLIPIDS sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) possess a wide spectrum of biological activities including stimulation of cell growth, inhibition of apoptosis, induction of actin cytoskeletal reorganization, and stimulation of cell migration (1–3, 7, 29). S1P interacts with five of eight "endothelial differentiation gene" (EDG) receptors renamed in accordance with International Union of Pharmacology Societies nomenclature as S1P1 (EDG1), S1P2 (EDG5), S1P3 (EDG3), S1P4 (EDG6), and S1P5 (EDG8). LPA interacts with LPA1 (EDG2), LPA2 (EDG4), and LPA3 (EDG7) receptors. S1P1, S1P2, and S1P3 receptors are widely distributed (1–3), whereas S1P4 receptors are confined to lymphoid and hematopoietic tissues (9), and S1P5 receptors are confined to oligodendrocytes (14).

Current understanding of G protein coupling of S1P receptors is based on receptor expression studies in cell lines. In cells expressing S1P1 receptors, S1P stimulates phosphoinositide (PI) hydrolysis, Ca2+ mobilization, and ERK1/2 in a pertussis toxin (PTx)-sensitive fashion, implying coupling to Gi/Go (27, 30); immunoprecipitation of S1P1 receptors in these cells leads to coprecipitation of G{alpha}i1 and G{alpha}i3 (16). In cells expressing S1P2 or S1P3 receptors, S1P stimulates PI hydrolysis and Ca2+ mobilization in both a PTx-sensitive and -insensitive fashion and activates ERK1/2 in a PTx-sensitive, Ras-dependent fashion (6, 30); in addition, S1P activates RhoA and induces stress fiber formation and cell migration (6). Analysis of G protein coupling suggests that S1P2, and probably S1P3 receptors, are coupled to Gi, Gq, and G12/G13 (6, 28). Rac1 is activated via S1P3 and inhibited via S1P2; Rac1 activation reflects predominant coupling of S1P3 to Gi, and Rac1 inhibition reflects predominant coupling of S1P2 to G12/G13 (28). Recent studies (9) suggest that S1P4 receptors regulate cell shape and motility via Gi and G12/G13. S1P5 is coupled to Gi/o and G12 but not to Gs or Gq (18).

Although cell lines transfected with individual S1P receptors can be a useful guide, G protein coupling in native cells may differ depending on the number of S1P receptors and/or the complement of G proteins expressed in these cells. Coronary arterial smooth muscle cells contract in response to S1P and express S1P2 receptors and, to a lesser extent, S1P3 and S1P1 receptors (26). Gastric smooth muscle cells also contract in response to S1P and express both S1P2 and S1P1 receptors (31). All the G proteins (Gq, Gi1, Gi2, Gi3, and G13) except for Gs are activated by S1P in gastric smooth muscle cells (31). Both Gq and Gi contribute to PI hydrolysis, Ca2+ release, and initial muscle contraction by activating G{alpha}q-dependent phospholipase (PLC)-beta1 and Gbeta{gamma}i-dependent PLC-beta3, resulting in inositol 1,4,5-trisphosphate (IP3)-stimulated Ca2+ release and activation of Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Both Gq and G13 contribute to sustained muscle contraction by activating RhoA/Rho kinase/PKC-dependent pathways that mediate inhibition of MLC phosphatase and activation of Ca2+-independent MLC kinase (20).

In the present study, we used molecular and pharmacological approaches to determine the functional contribution of each receptor type. Lentiviral vectors encoding small interfering RNAs (siRNAs) were transiently transfected into cultured smooth muscle cells to silence S1P1 or S1P2 receptors. In addition, S1P2 receptors were chemically inactivated after protection of S1P1 receptors with a selective S1P1 receptor agonist. The results indicate that only S1P2 receptors activate PLC-beta- and RhoA-dependent pathways that mediate initial and sustained contraction, respectively.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
siRNA for S1P1 and S1P2 receptors. Lentiviral vector constructs encoding enhanced green fluorescent protein (EGFP) as an internal marker together with siRNA for S1P1 or S1P2 receptors were generated using a modified two-step PCR-based strategy (8, 11). Briefly, three siRNA expression cassettes for each receptor were generated through consequential two rounds of PCR and individually cloned into pLL3.7 lentiviral vector via XbaI/XhoI cloning sites. The sequence of each siRNA cassette was confirmed by restriction enzyme digestion with BamHI/EcoRI and DNA sequencing. Silencing efficiency and specificity of these siRNA constructs were determined by Western blot and immunocytochemical studies in human embryonic kidney (HEK)-293T cells and RT-PCR analysis in gastric smooth muscle cells (11). The most effective siRNA constructs, S1P1b, and S1P2a, were used in the present study. The sequences for S1P1b and S1P2a were 5'-GAAGACCTGTGACATCCTGTA-3' and 5'-ACCAAGGAGACGCTGGACATG-3'.

Lentiviral vector transfection into cultured smooth muscle cells. Dispersed gastric smooth muscle cells were prepared from the circular muscle layer of the rabbit distal stomach by sequential enzymatic digestion, filtration, and centrifugation as previously described (19, 21, 31). The cells were cultured to confluence in DMEM with 10% FBS plus antibiotics. Cells in first passage grown on six-well plates were transiently transfected with lentiviral vector encoding siRNA for S1P1 or S1P2 receptors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Lentiviral vector (2 µg) with or without siRNA in 125 µl Opti-MEM medium was mixed with 5 µl Lipofectamine 2000 in 125 µl Opti-MEM. The mixture was incubated at room temperature for 20 min and added to wells containing 1.5 ml DMEM-10% FBS for 1 day. The medium was then replaced with DMEM-10% FBS plus antibiotics for 2 days. The cells were maintained for a final 24 h in DMEM without FBS before experiments were started. Fluorescence analysis of EGFP showed a transfection efficiency of 60–70%.

G protein activation assay. Activation of specific G proteins was determined by measuring the increase in G{alpha} binding to guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) on addition of agonist (S1P) as previously described (22, 31). Cells obtained 3 days after transfection with lentiviral siRNA vectors were homogenized in a medium consisting of 20 mM HEPES (pH 7.4), 2 mM MgCl2, 1 mM EDTA, and 2 mM 1,4-dithiothreitol (DTT). The homogenate was centrifuged at 30,000 g for 30 min at 4°C, and the membranes were solubilized at 4°C in 20 mM HEPES (pH 7.4) buffer containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized membranes were incubated at 37°C for 20 min in a medium containing 100 nM [35S]GTP{gamma}S and 10 mM HEPES (pH 7.4) in the presence or absence of S1P. Ten volumes of 100 mM Tris·HCl (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP were used to stop the reaction. The membranes were incubated for 2 h on ice in wells separately coated with antibodies to G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}q, and G{alpha}13. After each well was washed with PBS, radioactivity was counted by liquid scintillation.

Assay for PLC-beta activity. PLC-beta activity was determined in smooth muscle from the formation of total inositol phosphates using ion-exchange chromatography as described previously (22). Cultured (after transfection) or freshly dispersed smooth muscle cells labeled with myo-[3H]inositol (0.5 µCi/ml) in inositol-free DMEM without FBS were washed with PBS and treated with S1P or S1P1 agonist (SEW2871; 1 µM) for 60 s in 1 ml of 25 mM HEPES medium (pH 7.4) containing (in mM) 115 NaCl, 5.8 KCl, 2.1 KH2PO4, 2 CaCl2, and 14 glucose. The reaction was terminated by the addition of 940 µl of chloroform-methanol-HCl (50:100:1). The samples were extracted with chloroform and water, and the phases were separated by centrifugation for 15 min at 1,000 g. The aqueous phase was applied to a DOWEX AG-1 column, and [3H]inositol phosphates were eluted with 0.8 M ammonium formamate-0.1 M formic acid. Radioactivity was determined by liquid scintillation and was expressed as counts per minute (cpm).

Assay for Rho kinase activity. Rho kinase activity was determined in cell extracts by immunokinase assay as described previously (24). Cultured (after transfection) or freshly dispersed smooth muscle cells were solubilized with lysis buffer containing 50 mM Tris·HCl (pH 7.5), 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin. An equal amount of protein extracts was incubated with Rho kinase-2 antibody plus protein A/G agarose overnight at 4°C. The immunoprecipitates were washed twice in a medium containing 10 mM MgCl2 and 40 mM HEPES (pH 7.4) and then incubated for 5 min on ice with 5 µg of myelin basic protein. The assay was initiated by the addition of 10 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol) and 20 µM ATP, followed by incubation for 10 min at 37°C. The 32P-labeled myelin basic protein was absorbed onto phosphocellulose disks, and free radioactivity was removed by repeated washings with 75 mM phosphoric acid. The amount of radioactivity on the disks was measured by liquid scintillation.

Selective protection of S1P1 receptors and inactivation of S1P2 receptors. A technique of selective preservation of one receptor type was used to determine the function of S1P1 and S1P2 receptors. This technique was extensively validated and used to determine the function of G protein-coupled receptors coexpressed in the same cell (e.g., 5-hydroxytryptamine receptors) (15, 19, 21, 23). The technique involves protection of one receptor type with a selective agonist or antagonist followed by inactivation of all unprotected receptors by brief treatment with a low concentration of N-ethylmaleimide. In the present study, the selective S1P1 receptor agonist SEW2871 was used to protect S1P1 and inactivate all other receptors including S1P2 receptors. S1P was used as a control to preserve both S1P1 and S1P2 receptors and inactivate all other receptors. Freshly dispersed muscle cells were incubated with SEW2871 or S1P at 31°C for 2 min followed by addition of 5 µM N-ethylmaleimide for 20 min. The cells were centrifuged twice at 150 g for 10 min to eliminate the protective agonist and N-ethylmaleimide and resuspended in fresh HEPES buffer. The initial and sustained contractile response of cells treated in this fashion was compared with the response of untreated (naive) cells. Previous studies (15, 19, 21, 23) have shown that the coupling of protected receptors to signaling pathways remains intact. Smooth muscle cells incubated with N-ethylmaleimide without protective ligand did not respond to agonists but retained their ability to respond to agents that bypass receptors (e.g., ionomycin, KCl, and forskolin), implying that postreceptor mechanisms were intact (5, 10).

Measurement of contraction in dispersed smooth muscle cells. Muscle cell contraction was measured in freshly dispersed muscle cells by scanning microscopy as described previously (19, 20). Cell aliquots containing ~104 muscle cells/ml were treated with S1P or the selective S1P1 receptor agonist SEW2871 in the presence or absence of the S1P1 antagonist VPC23019; the reaction was terminated with 1% acrolein. The lengths of muscle cells treated with S1P or SEW2871 were compared with the lengths of untreated cells, and contraction was expressed as the percent decrease in cell length from control.

Statistical analysis. The results are expressed as means ± SE of n experiments and were analyzed for statistical significance using Student's t-test for paired or unpaired values.

Materials. S1P was obtained from BioMol Research Labs (Plymouth Meeting, PA), SEW2871 from Calbiochem (San Diego, CA), (R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester (VPC23019) from Avantis Polar Lipids (Alabaster, AL), [{gamma}-32P]ATP from Amersham Pharmacia Biotech (Piscataway, NJ), and myo-[3H]inositol from DuPont New England Nuclear (Boston, MA). G{alpha} and Rho kinase-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All the other reagents were from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distinctive patterns of G protein activation by S1P1 and S1P2 receptors in smooth muscle. Previous studies (31) have demonstrated that S1P activates G{alpha}q, G{alpha}13, and all three isoforms of G{alpha}i in gastric smooth muscle cells. Because these cells express both S1P1 and S1P2 receptors, it was not possible to determine the pattern of G protein activation by each receptor. In the present study, we measured G protein activation in response to S1P after selective silencing of S1P1 or S1P2 receptors in cultured gastric smooth muscle cells transiently transfected with lentiviral vectors encoding siRNAs for each receptor. Silencing of S1P1 receptors abolished S1P-stimulated activation of G{alpha}i3 and inhibited activation of G{alpha}i1 by ~60% but had no effect on activation of G{alpha}i2, G{alpha}q, and G{alpha}13 (Fig. 1). In contrast, silencing of S1P2 receptors abolished S1P-stimulated activation of G{alpha}q, G{alpha}13, and G{alpha}i2 and inhibited activation of G{alpha}i1 by ~40% but had no effect on activation of G{alpha}i3 (Fig. 1). Thus S1P1 receptors are coupled to Gi3 and Gi1, whereas S1P2 receptors are coupled to Gq, G13, Gi2, and Gi1. Only Gi1 activation is shared by both receptors.


Figure 1
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  		Fig. 1. Distinct patterns of G protein activation by sphingosine-1-phosphate (S1P)1 and S1P2 receptors. Cultured gastric smooth muscle cells (SMCs) in first passage were transfected with lentiviral vectors encoding small interfering RNA (siRNA) for S1P1 or S1P2 receptors or empty vector (control). After 3 days, G protein activity was determined from the difference in the binding of specific G{alpha} subunits to 0.1 µM [35S]GTP{gamma}S alone or in the presence of 1 µM S1P; the difference for each G{alpha} subunit is shown by the open bars and expressed as counts per minute (cpm). Binding of GTP{gamma}S alone to G{alpha} subunits ranged from 852 ± 47 to 1,034 ± 65 cpm. Silencing of S1P2 receptors abolished S1P-stimulated G{alpha}q, G{alpha}13, G{alpha}i2 activity and partially inhibited G{alpha}i1 activity (~40%). Silencing of S1P1 receptors abolished G{alpha}i3 activity and partially inhibited G{alpha}i1 activity (~60%). Values are means ± SE of 4 experiments. *P < 0.05 and **P < 0.01, significant differences from control G{alpha} binding activity in response to S1P (shown by open bars for each G{alpha} subunit).

 
Silencing of S1P2 receptors suppresses S1P-induced activation of PLC-beta and Rho kinase in smooth muscle. We have previously shown that in smooth muscle cells expressing both receptors, S1P stimulated PLC-beta activity in both a PTx-sensitive and -insensitive fashion, implying participation of both Gq and Gi (31). The involvement of both G proteins was confirmed by expression of G{alpha}q or G{alpha}i minigenes to inactivate Gq or all isoforms of Gi. Silencing of S1P2 receptors abolished S1P-stimulated PLC-beta activity (measured at 60 s), whereas silencing of S1P1 receptors had no effect (Fig. 2). In light of our previous study (31), this implied that activation of PLC-beta was mediated by Gq (PTx-insensitive component) and by Gi2 and/or Gi1 (PTx-sensitive component) but not by Gi3, which is exclusively coupled to S1P1 receptors (Fig. 1).


Figure 2
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  		Fig. 2. Silencing of S1P2 receptors abolishes S1P-induced activation of phospholipase C (PLC)-beta. Cultured gastric SMCs in the first passage were transfected with lentiviral vectors encoding siRNA for S1P1 or S1P2 receptors or empty vector (control). After 2 days, cells were labeled with myo-[3H]inositol and then treated with S1P (1 µM) or vehicle for 60 s. [3H]inositol phosphate was determined as described in MATERIALS AND METHODS and expressed as cpm. Values are means ± SE of 3 experiments. **P < 0.01, significant increase in inositol phosphate production above basal level (vehicle) in control cells and cells expressing S1P1-siRNA. No significant (ns) increase in inositol phosphate production in cells expressing S1P2-siRNA was found.

 
We have also previously shown that in smooth muscle cells expressing both receptors, S1P-stimulated Rho kinase activity was partially inhibited by expression of G{alpha}q or G{alpha}13 minigenes and virtually abolished by coexpression of both minigenes, implying that RhoA was activated by both Gq and G13 (31). In the present study, silencing of S1P2 receptors abolished S1P-stimulated Rho kinase activity (measured at 5 min), whereas silencing of S1P1 receptors had no effect (Fig. 3). The involvement of S1P2 receptors reflected their ability to activate Gq and G13 (Fig. 1).


Figure 3
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  		Fig. 3. Silencing of S1P2 receptors abolishes S1P-induced activation of Rho kinase. Cultured gastric SMCs in first passage were transfected with lentiviral vectors encoding siRNA for S1P1 or S1P2 receptors or empty vector (control). After 3 days, cells were treated with S1P (1 µM) or vehicle for 5 min, and Rho kinase activity was determined by immunokinase assay as described in MATERIALS AND METHODS and expressed as cpm. Values are means ± SE of 3 experiments. **P < 0.01, significant increase in Rho kinase activity above basal level (vehicle) in control cells and cells expressing S1P1-siRNA. No significant increase in Rho kinase activity in cells expressing S1P2-siRNA was found.

 
Chemical inactivation of S1P2 receptor suppresses S1P-induced smooth muscle contraction. To corroborate the results obtained by selective receptor silencing, we compared the contractile responses to S1P and a selective S1P1 receptor agonist, SEW2871, and used the latter to protect S1P1 receptors and chemically inactivate S1P2 receptors. Treatment of freshly dispersed smooth muscle cells with SEW2871 did not elicit contraction, whereas treatment with S1P caused an initial contraction (measured at 30 s) and a sustained contraction (measured at 5 min; Fig. 4A).


Figure 4
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  		Fig. 4. Chemical inactivation of S1P2 receptors abolishes S1P-induced smooth muscle contraction. A: freshly dispersed SMCs were treated with the selective S1P1 receptor agonist SEW2871 (1 µM) or S1P (1 µM) for 30 s to determine initial contraction and for 5 min to determine sustained contraction. Cell length was measured by scanning microscopy and contraction was expressed as the %decrease in cell length from control. Values are means ± SE of 3–4 experiments. B: dispersed SMCs were incubated with SEW2871 (1 µM) or S1P (1 µM) for 2 min, after which 5 µM N-ethylmaleimide was added and incubation was maintained for 20 min to inactivate unprotected receptors. After the SMCs were washed, the initial and sustained contractile responses to S1P (1 µM) were measured as described above. Selective inactivation of S1P2 receptors after protection of S1P1 receptors with SEW2871 abolished the contractile response to S1P, whereas protection of S1P1 and S1P2 receptors after protection with S1P preserved the contractile response to S1P. Values are means ± SE of 3–4 experiments.

 
Treatment of the cells with SEW2871 for 2 min to protect S1P1 receptors followed by the addition of N-ethylmaleimide to inactivate all unprotected receptors including S1P2 receptors abolished the contractile response to S1P (Fig. 4B). In contrast, treatment of the cells with S1P protected both S1P1 and S1P2 receptors and preserved the response to S1P (Fig. 4B). Studies to protect S1P2 receptors and inactivate S1P1 receptors could not be done because the S1P2 receptor antagonist JTE013 reported in other studies (13, 26) is not commercially available.

Selective S1P1 receptor agonists do not activate PLC-beta or Rho kinase. Treatment of freshly dispersed muscle cells with S1P stimulated both PLC-beta and Rho kinase activities, measured at 60 s and 5 min, respectively, whereas treatment of the cells with SEW2871 had no effect (Fig. 5). S1P-stimulated PLC-beta and Rho kinase activities were not affected by pretreatment of the cells with VPC23019, a mixed S1P1/S1P3 antagonist (Fig. 5) (4). The results confirmed that S1P1 receptors do not activate pathways that mediate initial and sustained muscle contraction.


Figure 5
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  		Fig. 5. A selective S1P1 receptor agonist does not activate PLC-beta or Rho kinase in freshly dispersed gastric SMCs. Cells were treated with a selective S1P1 receptor agonist SEW2871 (1 µM) or S1P (1 µM) for 60 s for measurement of PLC-beta activity (A) or for 5 min for measurement of Rho kinase activity (B). Activities were determined as described in MATERIALS AND METHODS and expressed as cpm. Pretreatment with the mixed S1P1/S1P3 receptor antagonist VPC23019 had no effect on the response to S1P. Values are means ± SE of 3 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of two or more S1P receptor types on target cells complicates the analysis of signaling pathways, in particular the assigning of function to specific receptors. Recent studies (31) have demonstrated coexpression of S1P1 and S1P2 receptors in smooth muscle cells of the gut. In these cells, S1P activates a full complement of PTx-sensitive and -insensitive G proteins and initiates signaling cascades that mediate Ca2+-dependent initial contraction and Ca2+-independent sustained contraction (24, 31). The initial contraction reflected activation of PLC-beta1 and PLC-beta3 by PTx-insensitive (Gq) and -sensitive (Gi) G proteins, respectively, whereas sustained contraction reflected activation of RhoA via PTx-insensitive G proteins (Gq and G13). In the present study, PLC-beta (PI hydrolysis) and Rho kinase activities were used as specific markers of the pathways that mediate initial and sustained contraction, respectively.

Molecular (selective receptor silencing by siRNA) and pharmacological (selective receptor inactivation) approaches were used to determine whether S1P1 and/or S1P2 receptors mediated these signaling pathways. The siRNA constructs for S1P2 and S1P1 receptors used in the present study efficiently and stably silence S1P2 and S1P1 receptors endogenously expressed in gastric smooth muscle cells (11). In these studies, the lentiviral vectors encoding each siRNA were delivered into smooth muscle cells by infection with lentivirus (11). In the present study, the same vectors were transiently transfected into smooth muscle cells using Lipofectamine 2000. Transient silencing of S1P2 but not S1P1 receptors abolished S1P-induced PLC-beta and Rho kinase activities (Figs. 2 and 3), confirming the results obtained on stable silencing of each receptor type by lentiviral infection (11).

Receptor silencing yielded a clear pattern of G protein activation by each receptor. S1P2 receptors activated Gq, G13, Gi2, and Gi1 (~40%), whereas S1P1 receptors activated Gi3 and Gi1 (~60%). Because S1P stimulated both PTx-sensitive and -insensitive PLC-beta activities as previously shown (31) and silencing of S1P2 receptors abolished PLC-beta activity, it follows that PLC-beta activity reflected activation of Gq (PTx-insensitive component) and Gi2 and/or Gi1 (PTx-sensitive component). It also follows that coupling of S1P1 receptors to Gi3 or Gi1 did not lead to activation of PLC-beta. The inability of S1P1 receptors to activate PLC-beta in smooth muscle cells contrasts with observations made in Chinese hamster ovary or human erythroleukemia cells, where expression of S1P1 receptors resulted in S1P-induced, PTx-sensitive stimulation of PI hydrolysis and Ca2+ mobilization (27). Expression of S1P1 receptors in insect Spodoptera frugiperda Sf9 or monkey kidney fibroblast (COS7) cells, however, did not result in Ca2+ mobilization (32). The contrasting results between various cell lines and between cell lines and native cells (e.g., smooth muscle cells) underline the importance of determining G protein coupling and signaling pathways in each cell type.

Similarly, because silencing of S1P2 receptors (Fig. 3) or coexpression of Gq and G13 minigenes (31) abolished Rho kinase activity, it follows that Rho kinase activity reflected activation of Gq and G13 by S1P2 receptors. The pattern is consistent with previous studies (25) showing that receptors coupled exclusively to Gi do not activate RhoA or Rho kinase.

The effect of receptor silencing on G protein activation and on specific markers of signaling pathways (PLC-beta and Rho kinase) implied that initial and sustained muscle contraction was mediated exclusively by S1P2 receptors. This conclusion was supported further by direct measurement of initial and sustained contraction in freshly dispersed smooth muscle cells. A selective S1P1 receptor agonist did not elicit contraction or stimulate PLC-beta and Rho kinase activities. When the agonist was used to protect S1P1 receptor so as to allow chemical inactivation of all other receptors including S1P2 receptors, the cells did not contract in response to S1P.

We have previously shown that some Gi-coupled receptors (e.g., opioid µ, {delta}, and {kappa}, somatostatin sstr3, and adenosine A1 receptors) are capable of mediating sustained contraction via sequential activation of PI 3-kinase and integrin-linked kinase (ILK) by Gbeta{gamma}i (12). ILK activates CPI-17, a potent endogenous inhibitor of MLC phosphatase, and acts as a Ca2+-independent MLC kinase, leading to sustained MLC20 phosphorylation and muscle contraction. This Gi-dependent mechanism did not contribute to sustained contraction mediated by S1P2 receptors, because sustained contraction induced by S1P (unlike initial contraction) was insensitive to PTx and was virtually abolished by a combination of G{alpha}q and G{alpha}13 antibodies (31).

The inability of Gi1/Gi3-coupled S1P1 receptors to elicit initial or sustained muscle contraction deserves further comment. Muscarinic M2 receptors, which activate PLC-beta3 but do not stimulate initial or sustained contraction, are preferentially coupled via Gbeta{gamma}i3 to sequential activation of PI 3-kinase, p21-activated protein kinase (PAK1), and p38-MAPK: PAK1 phosphorylates and inactivates MLC kinase, whereas p38-MAPK phosphorylates and inactivates ILK, thus precluding phosphorylation of MLC20 and smooth muscle contraction (12). It is possible that a mechanism involving inactivation of ILK by p38 MAPK underlies the inability of S1P1 (or S1P2) receptors to elicit Gbeta{gamma}i-dependent sustained contraction. A more plausible mechanism, however, is akin to that identified recently for cannabinoid CB1 receptors in gastric smooth muscle cells: these receptors are coupled to an atypical G protein in which the G{gamma}-like domain of RGS6 (regulator of G protein signaling) binds to Gbeta5 and G{alpha}i2 (17). On dissociation, the RGS6-Gbeta5 complex, unlike a typical Gbeta{gamma}i dimer, does not activate downstream effector enzymes such as PLC-beta3 or PI 3-kinase and thus does not initiate signaling cascades capable of stimulating initial or sustained contraction. This aspect will be explored in future studies.

We have previously shown that smooth muscle cells express two isoforms of sphingosine kinase (SPK1 and SPK2); these enzymes phosphorylate sphingosine, a metabolic product of ceramide, to yield S1P (31). The kinases are activated by various growth factors, cytokines, and G protein-coupled receptor agonists (e.g., acetylcholine). S1P formed within smooth muscle cells may be transported to the cell surface to activate S1P1 and S1P2 receptors and modulate the response to contractile agonists, growth factors, or cytokines. The functional significance of S1P in smooth muscle under physiological or pathological conditions remains to be explored.

In summary, the specific functions of S1P1 and S1P2 receptors coexpressed in smooth muscle of the gut were characterized by selective receptor silencing with siRNA and by selective chemical inactivation. Each receptor was shown to couple to a distinct complement of G proteins, and only S1P2 receptors were shown to activate signaling pathways that mediate initial and sustained muscle contraction.


   ACKNOWLEDGMENTS
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15564.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Hu, Dept. of Physiology, P.O. Box 980551, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (e-mail: whu{at}vcu.edu)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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