Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

Huiping Zhou and Karnam S. Murthy

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298

Submitted 6 October 2003 ; accepted in final form 28 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC20) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P1 and S1P2 receptors. S1P activated Gq, G13, and all Gi isoforms and stimulated PLC-{beta}1, PLC-{beta}3, and Rho kinase activities. PLC-{beta} activity was partially inhibited by pertussis toxin (PTX), G{beta} or G{alpha}q antibody, PLC-{beta}1 or PLC-{beta}3 antibody, and by expression of G{alpha}q or G{alpha}i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G{alpha}13 or G{alpha}q minigene and abolished by expression of both. S1P stimulated Ca2+ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC50 1 nM). Initial contraction and MLC20 phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G{alpha}q or G{beta} antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC20 phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P2 and S1P1 involving concurrent activation of PLC-{beta}1 and PLC-{beta}3 via G{alpha}q and G{beta}{gamma}i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca2+ release and MLCK-mediated MLC20 phosphorylation, and 2) sustained contraction exclusively mediated by S1P2 involving activation of RhoA via G{alpha}q and G{alpha}13, resulting in Rho kinase- and PKC-dependent MLC20 phosphorylation.

muscle contraction; signal transduction


THE BIOACTIVE LYSOPHOSPHOLIPID sphingosine 1-phosphate (S1P) has been implicated in a variety of biological processes, such as cell growth and differentiation, cell survival, and cell migration (1, 4, 15, 16, 38). S1P is formed by phosphorylation of sphingosine, a metabolic product of ceramide, which is derived from membrane-bound sphingomyelin or formed de novo by condensation of serine and palmitoyl-CoA (32, 33). Sphingosine phosphorylation is mediated by sphingosine kinase (SPK); two isoforms of this enzyme, SPK1 and SPK2, have been cloned and characterized in human, mouse, yeast, and plant (12, 14). Intracellular levels of S1P are regulated by SPK and by S1P-specific phosphatase and lyase (13, 30, 33). Basal intracellular levels of S1P are low but can increase rapidly although transiently on activation of SPK by various agents, including growth factors (e.g., platelet-derived growth factor, nerve growth factor, and epidermal growth factor), cytokines, and G protein-coupled receptor agonists such as muscarinic and purinergic agonists (10, 18, 31, 32). Potential phosphorylation sites have been identified on SPK1 and/or SPK2 for protein kinase (PK)C, PKA, and tyrosine kinase(s), suggesting the possibility of regulatory phosphorylation.

S1P was viewed until recently as an intracellular messenger, although a specific intracellular target could not be identified. More recent studies have focused on the ability of S1P to interact specifically with several members of the endothelial differentiation gene (EDG) family of G protein-coupled receptors (9, 11, 3133, 35). Most responses attributed to the intracellular action of S1P could be reproduced by exposure of the cells to low concentrations of extracellular S1P (4, 17, 27), whereas gene disruption of specific S1P receptors in mice led to failure of vascular maturation, cell migration, or cardiac development (15). There is evidence that S1P synthesized in cells is transported to the extracellular space via a yet unspecified transport system, presumed to be akin to the cystic fibrosis transport protein (2). S1P is abundantly produced by platelets and could reach endothelial and other cells via the circulation bound to plasma proteins (38). Thus S1P could gain access to the receptors via autocrine/paracrine or quasiendocrine pathways. The effect of muscarinic, purinergic, or other G protein-coupled receptor agonists capable of activating SPK could be potentiated on release of S1P to act on S1P receptors (18, 31, 32).

Five of eight EDG receptors are selectively activated by S1P and have been renamed S1P receptors in accordance with International Union of Pharmacology Societies (IUPHAR) nomenclature. S1P1, S1P2, S1P3, S1P4, and S1P5 correspond, respectively, to EDG1, EDG5, EDG3, EDG6, and EDG8. EDG2, EDG4, and EDG7 are specifically activated by lysophosphatidic acid (LPA), a structurally related lysophospholipid, and have been renamed LPA receptors (LPA1, LPA2, and LPA3) (11, 3133).

S1P1, S1P2, and S1P3 receptors are widely expressed, whereas S1P4 is confined to lymphoid and hematopoietic cells and S1P5 to glial cells (11, 3133). On the basis of receptor expression studies in cell lines, S1P receptors appear to be coupled to distinctive complements of G proteins (11, 31, 36). S1P1 is coupled to Gi and to activation of the monomeric G protein Rac; S1P2 and S1P3 are coupled to Gi, Gq, G12/13, and RhoA; in addition, S1P2 is coupled to inhibition and S1P3 to activation of Rac (28). S1P4 and S1P5 are coupled to Gi and, in some cells, to G12/13 and RhoA (29, 32). S1P receptors activate different isoforms of phospholipase C (PLC)-{beta}, and except for S1P5, they also activate extracellular signal-regulated protein kinases (ERK1/2) (16, 37). Coupling of S1P receptors to specific G proteins or effector enzymes in native cells may differ depending on the number of S1P receptor types and the complement of G proteins expressed in these cells (3133, 36).

Little is known of the expression of S1P receptors or the signal transduction pathways initiated by these receptors in visceral and vascular smooth muscle. A recent study showed that S1P1, S1P2, and S1P3 receptors are expressed in human coronary arterial smooth muscle cells (27). When placed in culture, the cells contracted in response to S1P and the contraction was reversed by Clostridium botulinum C3 exoenzyme, which selectively inactivates RhoA (27). In the present study, we examined the expression of S1P receptors and SPK isoforms in freshly dispersed and cultured gastric smooth muscle cells and identified the signaling pathways initiated by these receptors. Selective G protein antibodies and minigene expression were used to identify the coupling of specific G proteins to effector enzymes, and selective inhibitors were used to characterize the pathways involved in initial and sustained 20-kDa myosin light chain (MLC20) phosphorylation and muscle contraction. The results demonstrated the coexpression of S1P1 and S1P2 receptors and both isoforms of SPK and identified distinct signaling pathways initiated by each receptor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of dispersed and cultured gastric smooth muscle cells. All procedures were conducted in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Smooth muscle cells were isolated from the circular muscle layer of rabbit distal stomach by sequential enzymatic digestion, filtration, and centrifugation as previously described (2225). The partly digested strips were washed, and muscle cells were allowed to disperse spontaneously for 30 min. Cells were harvested by filtration through 500-µm Nitex and centrifuged twice at 350 g for 10 min. For permeabilization, dispersed smooth muscle cells were treated for 5 min with saponin (35 µg/ml) and resuspended in low-Ca2+ (100 nM) medium as previously described (23). In some experiments, the cells were cultured in DMEM containing 10% fetal bovine serum until they attained confluence and were then passaged once for use in various studies (25).

RT-PCR analysis of S1P receptors and SPK isoforms. Specific primers were designed based on homologous sequences in human, rat, and mouse cDNAs for S1P1, S1P2, S1P3, S1P4, S1P5, SPK1, and SPK2. The sequences of the primers are listed in Table 1. Total RNA (5 µg) isolated from cultured gastric smooth muscle cells was reversibly transcribed and amplified by PCR under standard conditions as described previously (25). The PCR products were separated by electrophoresis in 1.2% agarose gel in the presence of ethidium bromide, visualized by ultraviolet fluorescence, and recorded by a ChemiImager 4400 fluorescence system. PCR products were purified and sequenced.


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Table 1. Sequences of PCR primers for S1P receptors and sphingosine kinase

 

Minigene construction and transfection into cultured smooth muscle cells. The cDNAs encoding the last COOH-terminal 11 amino acids of mouse G{alpha}q and G{alpha}13 and human G{alpha}i were amplified by PCR under standard conditions with Taq DNA polymerase (25). The 5'-end of sense primers contained a BamHI site followed by the ribosome binding consensus sequence (5'-GCCGCCACC-3'), a methionine (ATG) start code, and a glycine (GGA) to protect the ribosome binding site during translation and the nascent peptide against proteolytic degradation. An EcoRI site was synthesized at the 5'-end of the antisense primers immediately after the stop codon (TGA) (68). The purified PCR products were subcloned into the mammalian expression vector pcDNA3.1(+). The oligonucleotide sequence corresponding to the COOH-terminal 11 amino acid residues of G{alpha}i in random order was synthesized and ligated into pcDNA 3.1(+) as a control minigene. All of the minigene constructs were verified by DNA sequencing. All G{alpha} minigene constructs used for transfection experiments were purified with an Endotoxin-Free Maxi-prep kit (Qiagen). The sequences of expressed minigene peptides are listed in Table 2.


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Table 2. Sequences of expressed G protein COOH-terminal minigene peptides

 

Cultured rabbit gastric smooth muscle cells were transiently transfected with minigene plasmid DNA with Effectene transfection reagent (Qiagen). Transfection efficiency was monitored by cotransfection of pGreen Lantern-1. Analysis by fluorescence microscopy showed that ~80% of the cells were transfected (25).

Identification of receptor-activated G proteins. Activation of specific G proteins was determined from agonist-induced increase in G{alpha} binding to guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) as previously described (23, 39). Cells were homogenized in 20 mM HEPES (pH 7.4) containing 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 (CHAPS). The solubilized membranes were incubated for 20 min at 37°C with 100 nM 35S-labeled GTP{gamma}S in 10 mM HEPES (pH 7.4) in the presence or absence of agonist. The reaction was stopped with 10 vols of 100 mM Tris·HCl (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the membranes were incubated for 2 h on ice in wells precoated with specific antibodies to G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}q, G{alpha}13, and G{alpha}s. After washing with phosphate buffer, the radioactivity from each well was counted by liquid scintillation.

Assay of PLC-{beta} activity. Inositol phosphates were measured as described previously with anion exchange chromatography (23, 39). Freshly dispersed smooth muscle cells were labeled for 4 h with myo-2-[3H]inositol (0.5 µCi/ml) in inositol-free HEPES medium. Muscle cells in culture were labeled for 24 h in inositol-free Dulbecco's modified Eagle's medium containing 0.5 µCi/well (6-well plate). The cells were washed with PBS and treated with S1P (1 µM) for 60 s in 1 ml of 25 mM HEPES buffer (pH 7.4) consisting of (in mM) 115 NaCl, 5.8 KCl, 2.1 KH2PO4, 2 CaCl2, 0.6 MgCl2, and 14 glucose. The reaction was terminated by addition of 940 µl of chloroform-methanol-HCl (50:100:1). After extraction with 340 µl of chloroform and 340 µl of H2O, the aqueous phase was applied to Dowex AG-1 columns; [3H]inositol phosphates were eluted, and radioactivity was determined in a liquid scintillation counter.

Assay for Rho kinase activity. Rho kinase activity was determined by immunokinase assay in cell extracts as described previously (25). Rho kinase immunoprecipitates were washed with phosphorylation buffer and incubated for 5 min on ice with 5 µg of myelin basic protein. Kinase assays were 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. [32P]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.

Assay for adenylyl cyclase activity. cAMP production was measured by radioimmunoassay as described previously (22, 23). Briefly, muscle cells were incubated for 10 min with forskolin (10 µM) in the presence of 100 µM isobutylmethylxanthine followed by addition of S1P for 60 s and the reaction was terminated with 6% trichloroacetic acid. cAMP was measured in duplicate, and the results are expressed as picomoles per milligram of protein.

Measurement of Ca2+ release. 45Ca2+ release was measured in permeabilized muscle cells as described previously (22). The cells were suspended in a medium containing 100 nM Ca2+, 10 µM antimycin, 10 µCi/ml 45Ca2+, 1.5 mM ATP, and an ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine kinase). Steady-state Ca2+ uptake was measured after incubation for 60 min. S1P (1 µM) or inositol 1,4,5-trisphosphate (IP3, 1 µM) was added, and Ca2+ release was determined after 30 s. 45Ca2+ release was expressed as the decrease in steady-state 45Ca2+ content.

Measurement of MLC20 phosphorylation. Phosphorylated MLC20 was determined by immunoblot analysis with a phospho-specific Ser19 antibody as described previously (25). Cell lysate proteins were resolved by SDS-PAGE and electrophoretically transferred onto polyvinylidine difluoride membranes. Membranes were incubated for 12 h with phospho-specific Ser19 MLC20 antibody and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. The bands were identified by enhanced chemiluminescence.

Measurement of contraction in dispersed smooth muscle cells. Muscle cell contraction was measured in freshly dispersed muscle cells by scanning micrometry as described previously (19, 20). A cell aliquot containing 104 muscle cells/ml was added to 0.1 ml of medium containing S1P, and the reaction was terminated with 1% acrolein. The effect of G protein antibodies was determined in permeabilized muscle cells after preincubation for 1 h with 10 µg/ml of each antibody separately or in combination. The lengths of muscle cells treated with S1P were compared with the lengths of untreated cells, and contraction was expressed as the decrease in mean cell length from control.

Materials. S1P was obtained from Biomol Research Labs (Plymouth Meeting, PA), [{gamma}-32P]ATP from Amersham Pharmacia Biotech (Piscataway, NJ), myo-[3H]inositol and [35S]GTP{gamma}S from DuPont NEN (Boston, MA), U-73122, ML-9, Y-27632, and bisindolylmaleimide from Calbiochem (San Diego, CA) and polyclonal antibodies to G{alpha}i1 G{alpha}i2, G{alpha}i3, G{alpha}13, G{alpha}s, and G{beta} from Santa Cruz Biotechnology (Santa Cruz, CA). DNA sequencing was done by the Virginia Commonwealth University Nucleic Acids Core Facility.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of S1P receptors and SPK isoforms. Specific primers for each S1P receptor type and both isoforms of SPK (SPK1 and SPK2) were designed based on the conserved sequences in human, rat, and mouse cDNAs (Table 1). Only S1P1 and S1P2 were detected by RT-PCR in cultures of gastric smooth muscle cells. S1P3, which is abundantly expressed in coronary smooth muscle cells (27), and S1P4 and S1P5 were absent (Fig. 1A). Both isoforms of SPK were also expressed in gastric smooth muscle cells (Fig. 1B). The isolated partial amino acid sequences of rabbit S1P1, S1P2, SPK1, and SPK2 were 80–90% similar to the corresponding amino acid sequences of human and rat.



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Fig. 1. Selective expression of sphingosine 1-phosphate (S1P)1 and S1P2 receptors and sphingosine kinase (SPK) isoforms. RNA isolated from cultured gastric smooth muscle cells was reversibly transcribed and cDNA was amplified by PCR with specific primers for S1P1, S1P2, S1P3, S1P4, and S1P5 (A) and SPK1 and SPK2 (B). Experiments were done in the presence (+) or absence (-) of reverse transcriptase (RT). Primers and PCR product sizes are listed in Table 1.

 

Identification of G proteins coupled to S1P1 and S1P2 in smooth muscle. Studies in cell lines suggest that all S1P receptors are coupled to Gi, but except in a rare instance (36), the specific Gi isoform(s) coupled to each receptor has not been identified. S1P caused a significant increase in the binding of [35S]GTP{gamma}S to G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}q, and G{alpha}13, but not to G{alpha}s, in solubilized smooth muscle cell membranes (Fig. 2A). Figure 2, B and C, shows the kinetics and concentration dependence of G{alpha}q activation by S1P. As shown for other agonists, G protein activation was rapid and was maintained for up to 30 min. On the basis of studies of S1P receptor expression in cell lines (11, 28, 31, 36), activation of G{alpha}q and G{alpha}13 was attributed to interaction of S1P with S1P2, whereas activation of G{alpha}i1, G{alpha}i2, or G{alpha}i3 could reflect interaction of S1P with S1P1 and/or S1P2.



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Fig. 2. Complement of G proteins activated by S1P in gastric smooth muscle. A: membranes isolated from freshly dispersed smooth muscle cells were incubated with 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP{gamma}S) in the presence or absence of S1P (1 µM) for 20 min in wells variously coated with G{alpha} antibodies. B and C: time course and concentration dependence of G{alpha}q activation by S1P. Results are expressed as the S1P-induced increase in [35S]GTP{gamma}S binding to specific G{alpha} antibodies. Values are means ± SE of 4 experiments.

 

Activation of PLC-{beta} and inhibition of adenylyl cyclase. S1P stimulated a fourfold increase in phosphoinositide (PI) hydrolysis (PLC-{beta} activity) in freshly dispersed muscle cells (Fig. 3, A and B). U-73122 abolished S1P-stimulated PLC-{beta} activity, whereas pretreatment of cells with pertussis toxin (PTX; 400 ng/ml) for 1 h inhibited PLC-{beta} activity by 38 ± 7% (Fig. 3A). Pretreatment of the permeabilized muscle cells for 1 h with a specific G{alpha}q antibody or a common G{beta} antibody inhibited PLC-{beta} activity by 61 ± 5% and 40 ± 4%, respectively, and pretreatment with both antibodies abolished PLC-{beta} activity (90 ± 7%) (Fig. 3B). Pretreatment of permeabilized muscle cells with PLC-{beta}1 antibody inhibited S1P-stimulated PI hydrolysis by 55 ± 6%, to the same extent as pretreatment with G{alpha}q antibody; pretreatment with PLC-{beta}3 antibody inhibited PI hydrolysis by 33 ± 4%, to the same extent as pretreatment with G{beta} antibody or PTX. Pretreatment with both PLC-{beta} antibodies abolished PI hydrolysis (Fig. 3B). As shown with other agonists in these cells (20, 23, 24), the pattern of PI hydrolysis reflected activation of PLC-{beta}1 via G{alpha}q and PLC-{beta}3 via G{beta}{gamma}i. Accordingly, G{alpha}q antibody, G{beta} antibody, and PTX partially inhibited PI hydrolysis, whereas a combination of both antibodies abolished PI hydrolysis.



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Fig. 3. Effects of inhibitors, G protein antibodies, and G{alpha} minigene expression on S1P-stimulated phospholipase C (PLC)-{beta} activity. A: dispersed muscle cells were incubated for 10 min with U-73122 or for 60 min with pertussis toxin (PTX; 400 ng/ml) and then treated with S1P (1 µM) for 60 s. B: dispersed muscle cells were permeabilized, incubated for 1 h with 10 µg/ml of antibodies (Abs) to G{alpha}q, G{beta}, or both G{alpha}q and G{beta}, and with antibodies to PLC-{beta}1, PLC-{beta}3, or both PLC-{beta}1 and PLC-{beta}3, and then treated with S1P for 60 s. C: cultured muscle cells expressing control minigene GiR, G{alpha}q, G{alpha}i, or both G{alpha}q and G{alpha}i minigenes were treated with S1P for 60 s. In all experiments, the cells were prelabeled with myo-[3H]inositol. Results are expressed as total [3H]inositol phosphate formation. Values are means ± SE of 4 experiments. **Significant inhibition from control (P < 0.01).

 

Further evidence for specific G protein coupling was obtained by expression of G{alpha} minigenes in cultured smooth muscle cells. The synthetic peptides corresponding to the COOH terminus of G{alpha} subunits selectively antagonize G protein activation by blocking receptor-G protein interaction (68). We generated minigene plasmid constructs that encode COOH-terminal peptide sequences of G{alpha}i, G{alpha}q, and G{alpha}13 (Table 2). A control minigene was also generated containing the COOH terminus of G{alpha}i in random order (GiR). S1P-stimulated PLC-{beta} activity (4-fold above basal level) was inhibited by 64 ± 4% and 37 ± 6% in membranes derived from cultured muscle cells expressing G{alpha}q and G{alpha}i minigenes, respectively (Fig. 3C), and was abolished in membranes derived from cultured muscle cells expressing both G{alpha}q and G{alpha}i minigenes (95 ± 4%).

It is worth noting that inhibition of PLC-{beta} activity in cultured muscle cells expressing Gi minigene (37%) was similar to inhibition of PLC-{beta} activity by PTX (38%) in freshly dispersed muscle cells and by G{beta} antibody (40%) or PLC-{beta}3 antibody (33%) in permeabilized muscle cells. Similarly, inhibition of PLC-{beta} activity in cultured muscle cells expressing G{alpha}q minigene (64%) was similar to inhibition of PLC-{beta} activity by G{alpha}q antibody (61%) or PLC-{beta}1 antibody (55%) in permeabilized muscle cells. The results suggest that S1P2 receptors, which activate both Gq and Gi, are the dominant effectors of PI hydrolysis in these cells.

S1P (1 µM) inhibited forskolin-stimulated cAMP (22 ± 3 pmol/mg protein above basal level) in freshly dispersed muscle cells by 58 ± 5%, and the inhibition was completely reversed by PTX (data not shown). S1P also inhibited forskolin-stimulated cAMP (18 ± 3 pmol/mg protein) in cultured muscle cells by 74 ± 8%. Forskolin-stimulated cAMP (19 ± 4 pmol/mg protein) was not affected in muscle cells expressing Gi minigene, but in these cells, S1P failed to inhibit cAMP formation (17 ± 2 pmol/mg protein; not significant).

S1P-stimulated Ca2+ release and muscle contraction. The effect of S1P and IP3 on sarcoplasmic Ca2+ release was examined in permeabilized smooth muscle cells. S1P (1 µM) stimulated Ca2+ release (32 ± 1% decrease in steady-state 45Ca2+ content in 30 s), which was partly inhibited by PTX (15 ± 2% decrease in 45Ca2+ content) and abolished by U-73122 or by heparin (Fig. 4). The potent inhibitory effects of U-73122 and heparin implied that Ca2+ release was IP3 dependent; partial inhibition by PTX implied that IP3 formation reflected activation of PLC-{beta} via both Gq and Gi. This notion was supported by the fact that a combination of S1P and IP3 did not cause a significant increase in Ca2+ release over that of each agent separately (Fig. 4).



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Fig. 4. S1P-induced Ca2+ release in smooth muscle. Ca2+ release was measured in permeabilized muscle loaded with 45Ca2+ as previously described (22). U-73122 (1 µM) and heparin (100 ng/ml) were added 10 min and PTX (400 ng/ml) 60 min before addition of S1P (1 µM). The effects of S1P and inositol 1,4,5-trisphosphate (IP3; 1 µM) were equal and were not additive. Results are expressed as the maximal decrease in steady-state 45Ca2+ content (3.2 ± 0.4 nmol/mg protein) 30 s after addition of S1P. Values are means ± SE of 4 experiments. **Significant inhibition of Ca2+ release (P < 0.01).

 

Consistent with its ability to simulate Ca2+ release, S1P caused contraction of dispersed gastric smooth muscle cells. As with other agonists (19, 25), S1P-induced contraction was characterized by an initial transient phase followed by a sustained phase (Fig. 5A). Muscle contraction was detectable at concentrations below 0.1 nM and was concentration dependent with an EC50 of 1 nM (Fig. 5B).



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Fig. 5. Biphasic time course and concentration dependence of S1P-induced contraction of dispersed smooth muscle cells. A: S1P (1 µM) was added to freshly dispersed gastric smooth muscle cells for various intervals. Contraction consisted of transient initial peak followed by a sustained contraction. B: S1P was added to rabbit gastric smooth muscle cells at concentrations ranging from 1 pM to 1 µM, and contraction was measured at 30 s. Contraction was measured by scanning micrometry and expressed in micrometers as decrease in cell length from control (104 ± 4 µm). Values are means ± SE of 3 experiments.

 

Pathways mediating S1P-induced initial muscle contraction and MLC20 phosphorylation. As shown in recent studies (19, 25), the initial transient contraction (~2 min) induced by agonists is Ca2+ dependent and reflects sequential activation of PLC-{beta}, IP3-dependent Ca2+ release, Ca2+/calmodulin-dependent activation of MLC kinase (MLCK), and phosphorylation of Ser19 on MLC20, leading to interaction of actin and myosin and muscle contraction (25). Consistent with this pathway, the initial (30 s) contraction induced by S1P was partly inhibited by PTX (57 ± 5%) and virtually abolished by U-73122 and the MLCK inhibitor ML-9 (Fig. 6A). The partial inhibition of contraction by PTX implied participation of both Gq and Gi in stimulation of PI hydrolysis (Fig. 3) and Ca2+ release (Fig. 4). Studies in permeabilized muscle cells confirmed this notion: S1P-induced contraction was partly inhibited by preincubation for 1 h with G{alpha}q antibody (64 ± 3%) and G{beta} antibody (45 ± 5%) and was virtually abolished by preincubation with both antibodies (Fig. 6B). Initial contraction was not affected by the PKC inhibitor bisindolylmaleimide or the Rho kinase inhibitor Y-27632.



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Fig. 6. Inhibition of S1P-induced initial 20-kDa myosin light chain (MLC20) phosphorylation and muscle contraction. A: freshly dispersed gastric smooth muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), or Y-27632 (1 µM) for 10 min or PTX (400 ng/ml) for 60 min and then treated with S1P (1 µM) for 30 s. B: dispersed muscle cells were permeabilized and incubated with antibodies (10 µg/ml) to G{alpha}q, G{beta}, or both G{alpha}q and G{beta} for 60 min and then treated with S1P for 30 s. C: freshly dispersed smooth muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), or Y-27632 (1 µM) for 10 min and then treated with S1P (1 µM). MLC20 phosphorylation was measured at 60 s with phospho-specific Ser19 MLC20 antibody. Muscle contraction was measured by scanning micrometry and expressed in micrometers as the decrease in cell length from control (98 ± 3 µm). Values are means ± SE of 3–4 experiments. **Significant inhibition of S1P-induced initial contraction (P < 0.01).

 

The pattern of initial MLC20 phosphorylation induced by S1P and its inhibition by various agents closely paralleled that of initial contraction. Initial MLC20 phosphorylation was abolished by U-73122 and ML-9 but was not affected by bisindolylmaleimide or Y-27632 (Fig. 6C).

Pathways mediating S1P-induced activation of Rho kinase and sustained muscle contraction and MLC20 phosphorylation. Unlike initial contraction, agonist-stimulated sustained contraction and MLC20 phosphorylation are Ca2+ independent and are mediated cooperatively by Rho kinase- and PKC-dependent inhibition of MLC phosphatase (25). Sustained PKC activity in smooth muscle is largely dependent on RhoA-mediated activation of phospholipase D (PLD), which yields phosphatidic acid as its primary product (21). Dephosphorylation of phosphatidic acid to diacylglycerol results in sustained activation of PKC (21).

We showed previously (25, 26) that contractile agonists (e.g., acetylcholine and cholecystokinin) sequentially activate G13 and RhoA, resulting in activation of Rho kinase and PLD. In cultured smooth muscle cells, S1P (1 µM) caused a 3.4-fold increase in Rho kinase activity within 5 min, which was inhibited by 31 ± 6% in cells expressing G{alpha}q minigene and 61 ± 5% in cells expressing G{alpha}13 minigene and abolished in cells coexpressing G{alpha}q and G{alpha}13 minigenes (Fig. 7); expression of Gi minigene had no effect on S1P-stimulated Rho kinase activity (data not shown). Thus, unlike other contractile agonists in smooth muscle (25, 26), S1P activated RhoA via both Gq and G13.



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Fig. 7. Inhibition of S1P-stimulated Rho kinase activity by G{alpha}q and G{alpha}13 minigenes. Cultured gastric smooth muscle cells expressing G{alpha}q, G{alpha}13, or both G{alpha}q and G{alpha}13 minigenes were treated with S1P (1 µM) for 300 s; expression of Gi minigene had no effect (data not shown). Rho kinase activity was measured as described in MATERIALS AND METHODS. Values are means ± SE of 4 experiments. **Significant inhibition from control S1P-stimulated Rho kinase activity (P < 0.01).

 

Sustained S1P contraction measured at 5 min in freshly dispersed muscle cells was partially inhibited by bisindolylmaleimide and Y-27632 and was abolished by a combination of both inhibitors (Fig. 8A). U-73122, ML-9, and PTX had no effect on sustained contraction. Sustained contraction in permeabilized muscle cells was partially inhibited by preincubation for 1 h with G{alpha}q antibody (38 ± 3%) and G{alpha}13 antibody (49 ± 4%) and abolished by preincubation with both antibodies (93 ± 43%) (Fig. 8B). These results further confirm the dependence of S1P-induced activation of RhoA on both Gq and G13.



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Fig. 8. Inhibition of S1P-induced sustained MLC20 phosphorylation and muscle contraction. A: freshly dispersed gastric smooth muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), or Y-27632 (1 µM) for 10 min or PTX (400 ng/ml) for 60 min and then treated with S1P (1 µM) for 300 s. B: dispersed muscle cells were permeabilized and incubated with antibodies (10 µg/ml) to G{alpha}q, G13, or both G{alpha}q and G13 for 60 min and then treated with S1P for 300 s. C: freshly dispersed smooth muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), or Y-27632 (1 µM) for 10 min and then treated with S1P (1 µM) for 300s. MLC20 phosphorylation was measured with phospho-specific Ser19 MLC20 antibody. Muscle contraction was measured by scanning micrometry and expressed as the decrease in cell length from control (98 ± 3 µm). Values are means ± SE of 3–4 experiments. **Significant inhibition of S1P-induced sustained contraction (P < 0.01).

 

The pattern of sustained MLC20 phosphorylation induced by S1P and its inhibition by various agents closely paralleled that of sustained muscle contraction. Sustained MLC20 phosphorylation was partly inhibited by bisindolylmaleimide and Y-27632 but was not affected by U-73122 or ML-9 (Fig. 8C).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study demonstrates the expression of S1P1 and S1P2 receptors in gastric smooth muscle and their ability to induce initial Ca2+-dependent and sustained Ca2+-independent smooth muscle contraction and MLC20 phosphorylation via receptor coupling to a distinctive complement of heterotrimeric and monomeric G proteins: Gq, all three isoforms of Gi, G13, and RhoA. Coupling to Rac, which is activated by S1P1 and inhibited by S1P2 in other cells, was not examined (28).

The initial Ca2+-dependent phase reflected stimulation of PI hydrolysis via G{alpha}q and G{beta}{gamma}i, resulting in IP3-dependent Ca2+ release, and MLCK-dependent MLC20 phosphorylation and muscle contraction. The evidence can be briefly summarized as follows. S1P-stimulated PI hydrolysis was partly inhibited by PTX in intact muscle cells, by G{alpha}q or G{beta} antibodies and PLC-{beta}1 or PLC-{beta}3 antibodies in permeabilized muscle cells, and by expression of inhibitory Gq or Gi minigene in cultured muscle cells. A combination of G{alpha}q and G{beta} antibodies, PLC-{beta}1 and PLC-{beta}3 antibodies, or coexpression of both minigenes abolished PI hydrolysis. S1P induced concentration-dependent initial contraction and stimulated U-73122- and heparin-sensitive Ca2+ release; both Ca2+ release and initial contraction were partly inhibited by PTX. The ability of PTX to inhibit PI hydrolysis, and thus Ca2+ release and muscle contraction, conformed to a well-established pattern of stimulation of PI hydrolysis by various Gi-coupled receptors (opioid µ, {delta}, and {kappa}, adenosine A1 and P2Y2, and NPR-C receptors) via G{beta}{gamma}i-mediated activation of PLC-{beta}3 (20, 23, 24, 39). Initial contraction and MLC20 phosphorylation were abolished by U-73122 and ML-9. S1P2 expressed in cell lines couples to Gq, Gi, and G13, whereas S1P1 couples exclusively to Gi. Accordingly, initial contraction and MLC20 phosphorylation appear to be predominantly dependent on activation of S1P2.

In contrast, S1P-induced sustained MLC20 phosphorylation and muscle contraction reflected activation of RhoA. Previous studies (26) in these cells showed that activation of RhoA by other contractile agonists (e.g., cholecystokinin) was mediated by G13. With S1P as agonist, however, RhoA activation was mediated by both Gq and G13, because Rho kinase activity was partly inhibited in cells expressing G13 and Gq minigenes and abolished in cells coexpressing both minigenes. Activation of RhoA by both G proteins has been observed in some cell lines, including human embryonic kidney 293T cells (3). Consistent with participation of both G proteins, sustained contraction in permeabilized muscle cells was partly inhibited by preincubation with G{alpha}q and G{alpha}13 antibodies and abolished by preincubation with both antibodies. As shown in a recent study (25), both Rho kinase and PKC act downstream of RhoA to inhibit MLC phosphatase via distinct, although cooperative, mechanisms, resulting in sustained MLC20 phosphorylation and muscle contraction. S1P-induced sustained contraction and MLC20 phosphorylation were inhibited by Y-27632 and bisindolylmaleimide; contraction was abolished by a combination of both inhibitors.

RhoA-dependent stimulation of sustained muscle contraction and MLC20 phosphorylation were largely, if not exclusively, mediated by S1P2. A contribution by S1P1 is unlikely because it is not coupled to G13 in other cells (28, 36). Furthermore, activation of RhoA via Gi is precluded by the fact that Rho kinase activity was not inhibited by expression of Gi minigene and sustained contraction was not affected by PTX.

Recent studies on coronary artery (27) and airways smooth muscle cells (34) in culture showed that S1P activates RhoA and induces a contraction that is blocked by C3 exoenzyme and Y-27632. The results in coronary smooth muscle are consistent with the predominant expression of S1P3 and S1P2 in these cells, and the probable activation of RhoA via G13, but they differed from the results obtained in the present study in that contraction was not affected by a PKC inhibitor. The studies in coronary and airways smooth muscle did not distinguish the pathways mediating the initial and sustained phases of contraction.

The present study did not address the significance of SPK1 and SPK2 expression in gastric smooth muscle or the putative intracellular effects of S1P. Preliminary measurements suggest that acetylcholine causes a rapid, transient activation of SPK, lasting ~5 min, mediated by SPK1 (Zhou H and Murthy KS, unpublished observations). The rapid formation of S1P is subject to intracellular degradation by specific phosphatase and lyase but could, if transported outside the cell, act cooperatively with acetylcholine to trigger signaling pathways akin to those described in this study (10). It seems unlikely that S1P could act intracellularly to stimulate muscle contraction; its ability to stimulate sarcoplasmic Ca2+ release (5) requires high concentrations (>10 µM), unlike the low concentrations capable of eliciting Ca2+ release and contraction via S1P receptors. There is evidence, however, that pathways mediating growth and apoptosis could be initiated by intracellular S1P (29).

In summary, gastric smooth muscle expresses S1P1 and S1P2 receptors and both SPK isoforms. S1P-induced initial Ca2+-dependent MLC20 phosphorylation and muscle contraction and sustained RhoA-dependent MLC20 phosphorylation and muscle contraction are predominantly mediated by S1P2 receptors via their ability to activate Gq, Gi, G13, and RhoA.


    ACKNOWLEDGMENTS
 
GRANTS

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: K. S. Murthy, Depts. of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298.

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.


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