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Originally published In Press as doi:10.1074/jbc.M610767200 on December 17, 2006

J. Biol. Chem., Vol. 282, Issue 8, 5814-5824, February 23, 2007
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LPA4/p2y9/GPR23 Mediates Rho-dependent Morphological Changes in a Rat Neuronal Cell Line*

Keisuke Yanagida{ddagger}, Satoshi Ishii{ddagger}§1, Fumie Hamano{ddagger}, Kyoko Noguchi{ddagger}, and Takao Shimizu{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, Faculty of Medicine, the University of Tokyo and the §Precursory Research for Embryonic Science and Technology (PRESTO) of Japan Science and Technology Agency, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, June 21, 2006 , and in revised form, December 15, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA) is a potent lipid mediator that evokes a variety of biological responses in many cell types via its specific G protein-coupled receptors. In particular, LPA affects cell morphology, cell survival, and cell cycle progression in neuronal cells. Recently, we identified p2y9/GPR23 as a novel fourth LPA receptor, LPA4 (Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606). To assess the functions of LPA4 in neuronal cells, we used rat neuroblastoma B103 cells that lack endogenous responses to LPA. In B103 cells stably expressing LPA4, we observed Gq/11-dependent calcium mobilization, but LPA did not affect adenylyl cyclase activity. In LPA4 transfectants, LPA induced dramatic morphological changes, i.e. neurite retraction, cell aggregation, and cadherin-dependent cell adhesion, which involved Rho-mediated signaling pathways. Thus, our results demonstrated that LPA4 as well as LPA1 couple to Gq/11 and G12/13, whereas LPA4 differs from LPA1 in that it does not couple to Gi/o. Through neurite retraction and cell aggregation, LPA4 may play a role in neuronal development such as neurogenesis and neuronal migration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA,2 1- or 2-acyl-sn-glycero-3-phosphate) is a naturally occurring bioactive lipid mediator that controls growth, motility, and differentiation (1). LPA plays important roles in many biological processes, such as brain development, oncogenesis, wound healing, and immune functions (2). The effects of LPA on target cells are mediated by activation of its specific G protein-coupled receptors (GPCRs). The LPA1 (3), LPA2 (4), and LPA3 (5) receptors are the major members of the endothelial differentiation gene (EDG) family that interact with LPA. Pharmacological studies suggest that both LPA1 and LPA2 couple to at least three types of G proteins, Gi/o, Gq, and G12/13, whereas LPA3 couples to Gi/o and Gq but not G12/13 (6). Depending on the functional coupling of a given LPA receptor to G proteins, LPA activates diverse signaling cascades involving phosphoinositide 3-kinase, phospholipase C, mitogen-activated protein kinase, Rho family GTPase, and adenylyl cyclase (2, 7).

LPA is present in the brain at relatively high levels compared with other organs (8, 9). LPA influences the cell morphology of several neuronal cell lines, neural progenitors, and primary neurons (10). It has also been reported that LPA affects electrophysiology, cell survival, and cell cycle progression in neuronal cells (10, 11). Targeted deletion of LPA1 in mice produces olfactory deficits (12) and a behavioral abnormality (13). Furthermore, the use of LPA1 knockouts revealed that LPA1 is involved in the initiation of neuropathic pain (14). Exposure of the developing cerebral cortex to LPA produces dramatic changes in the folding of the brain, which do not occur in LPA1 and LPA2 double knockouts (15). However, the LPA receptor subtypes responsible for some neuronal effects have not been identified (16-18).

Recently, we identified p2y9/GPR23 as a fourth LPA receptor (LPA4) that is structurally distinct from the three LPA receptors of the EDG family (19). The expressed sequence tag cDNA encoding LPA4 was originally isolated from human brain (20, 21), and LPA4 expression has been detected in rat embryonic hippocampal neurons (22) and immortalized hippocampal progenitor cells (18). These facts suggest that LPA4 may have important roles in neurodevelopmental processes such as neurogenesis and neuronal migration. However, only very limited information is available regarding its physiological and biological functions. To assess the functional roles of LPA4 in neuronal cells, we generated B103 cells stably expressing LPA4. This study demonstrates that treatment of the LPA4-expressing cells with LPA leads to morphological changes, including cell rounding and cadherin-dependent cell adhesion following cell aggregation, both of which are mediated by the Rho/Rho-associated kinase (ROCK) pathway. The effects of LPA4 on the morphology of the neuronal cells were clearly distinct from those of LPA1, probably because LPA4 does not couple to Gi/o.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—B103 rat neuroblastoma cells were kindly provided by Dr. J. Chun (The Scripps Research Institute, La Jolla, CA). B103 cells expressing each of the LPA receptors were maintained on poly-L-lysine-coated 100-mm dishes (Iwaki, Tokyo, Japan) in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) and 0.3 mg/ml G418 (Wako, Osaka, Japan). For some experiments, cells were pretreated with 100 ng/ml pertussis toxin (PTX) (List Biological Laboratories, Campbell, CA; from a 400 µg/ml stock in 10 mM Tris-HCl (pH 7.4) and 2 M urea stored at 4 °C) for 12 h, 5 µM YM-254890 (a novel Gq/11 inhibitor (23), a kind gift from Dr. J. Takasaki, Astellas Pharma Inc., Tokyo, Japan; from a 10 mM stock in dimethyl sulfoxide (Sigma) stored at -30 °C) for 10 min, or 5 µM Y-27632 (Calbiochem; froma5mM stock in water stored at -30 °C) for 10 min. Pretreatment with vehicles of PTX and YM-254890 was used as a control.

Stable Expression of LPA1 and LPA4—A DNA fragment containing the entire open reading frame of LPA1 (NCBI accession number NM_001401 [GenBank] ) was first amplified from a cDNA prepared from human brain poly(A)+ RNA (Clontech) by PCR using Pfu turbo DNA polymerase (Stratagene, La Jolla, CA) and oligonucleotides (sense primer, 5'-AAGAAAATTTGTCTCCCGTAGCTCT-3' and antisense primer, 5'-CATGAGTTGACTTTTCTCCTCTCTC-3'). The entire open reading frame of LPA1 with an additional sequence encoding a hemagglutinin (HA) epitope (YPYDVPDYA) at the 5'-end was subsequently amplified from the resultant PCR products using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) and oligonucleotides (sense primer containing the KpnI and HA tag sequences, 5'-GGGGTACCGCCATGTACCCCTACGACGTGCCCGACTACGCCGCTGCCATCTCTACTTCC-3' and antisense primer containing the SpeI sequence, 5'-GGACTAGTCTAAACCACAGAGTGGTCATT-3'). The resultant DNA fragment was digested with KpnI and SpeI and subsequently cloned into the mammalian expression vector pCXN2.1, a slightly modified version of pCXN2 (24) with multiple cloning sites, between the KpnI and SpeI sites. HA-tagged human LPA4 cDNA was constructed and cloned into pCXN2.1 as described previously (19). B103 cells were transfected using the Lipofectamine 2000 reagent (Invitrogen). After 48 h, the transient expression of the HA epitope on the cell surface was confirmed by flow cytometric analysis (EPICS XL, Beckman Coulter, Fullerton, CA) with the 3F10 rat monoclonal anti-HA antibody (Roche Applied Science) and phycoerythrin-labeled anti-rat IgG (Beckman Coulter) as the secondary antibody. Stable transfectants were selected with 1 mg/ml G418 for 26 days. After staining the drug-resistant cells as described above, a group of HA-positive cells was sorted by flow cytometry (EPICS ALTRA, Beckman Coulter) and maintained with 0.3 mg/ml G418. Three weeks later, a second round of sorting was performed; the twice-immunopurified cells were used for experiments (termed B103-LPA1 and B103-LPA4 cells).

Binding Assay—Binding assay was done essentially as described previously (19), with minor modifications. Cells (4 x 106) were seeded in collagen-coated 100-mm plates (Iwaki), followed by 24 h of serum starvation. The cells were washed with phosphate-buffered saline (PBS) twice and scraped off. After further washing with binding buffer (25 mM HEPES-NaOH (pH 7.4), 10 mM MgCl2, and 0.25 M sucrose), the cells were suspended in the buffer with additional protease inhibitor mixture (Complete, Roche Applied Science), sonicated three times at 15 watts for 30 s, and centrifuged at 800 x g for 10 min at 4 °C. The supernatant was further centrifuged at 105 x g for 60 min at 4 °C, and resultant pellet was homogenized in ice-cold binding buffer. Binding assays were performed in 96-well plates in triplicate. 20 µg each of the membrane fractions from the twice-immunopurified cells was incubated in binding buffer containing 0.25% bovine serum albumin (BSA) (fatty acid-free, very low endotoxin grade; Serologicals Proteins, Kankakee, IL) with 2-fold serial dilutions (50-3.125 nM) of [3H]LPA (1-oleoyl[oleoyl-9, 10-3H]LPA, 57 Ci/mmol; PerkinElmer Life Sciences) for 60 min at 4 °C. The bound [3H]LPA was collected onto a Unifilter-96-GF/C (PerkinElmer Life Sciences) using a MicroMate 196 harvester (Packard Instrument Co.). The filter was then rinsed 10 times with ice-cold binding buffer and dried for 12 h at 50 °C. 25 µl of MicroScint-0 scintillation mixture (PerkinElmer Life Sciences) was added per well. The radioactivity that remained on the filter was measured with TopCount microplate scintillation counter (Packard Instrument Co.). Total and nonspecific bindings were evaluated in the absence and presence of 10 µM unlabeled LPA [1-oleoyl (18:1)-LPA; Cayman Chemical, Ann Arbor, MI), respectively. The specific binding value (disintegrations/min) was calculated by subtracting the nonspecific binding value (disintegrations/min) from the total binding value (disintegrations/min). A dissociation constant (Kd) and a maximum binding capacity (Bmax) were calculated by Scatchard analysis. Bmax and Kd values for B103-LPA1 cells were 0.8 pmol/mg protein and 18 nM, respectively. Those for B103-LPA4 cells were 6.0 pmol/mg protein and 58 nM. No specific binding was observed in vector-transfected B103 cells (B103-vector cells).

cAMP Measurement—Cells (3.2 x 104) were seeded in collagen-coated 96-well plates (Iwaki), followed by 24 h of serum starvation. To determine whether LPA receptors mediate the inhibition of adenylyl cyclase, an AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) was used as recommended in the manufacturer's instructions. The cells were washed twice with buffer A (Hanks' balanced salt solution (HBSS) containing 25 mM HEPES-NaOH (pH 7.4) and 0.1% BSA (Serologicals Proteins)) and incubated in 100 µl of buffer A containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX)) (from a 20 mM stock in dimethyl sulfoxide stored at -30 °C) (Sigma) for 15 min at room temperature. The reaction was initiated by adding 50 µl of various concentrations of LPA in buffer A with 50 µM forskolin (Wako; from a 10 mM stock in dimethyl sulfoxide stored at -30 °C). After 30 min of incubation at room temperature, the reaction was terminated by adding 16.6 µl of 10% Tween 20, followed by overnight storage at 4 °C. After centrifugation at 800 x g for 5 min, the cAMP concentration in the supernatant was measured in quadruplicate with a fusion system (PerkinElmer Life Sciences). To determine whether LPA receptors mediate the stimulation of adenylyl cyclase, the cAMP Biotrak EIA system (Amersham Biosciences) was used as recommended in the manufacturer's instructions. The cells were washed twice with HEPES-Tyrode's buffer (25 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 0.49 mM MgCl2, 12 mM NaHCO3, 0.37 mM NaH2PO4, and 5.6 mM D-glucose) containing 0.1% BSA (HEPES-Tyrode's BSA buffer) and incubated in 100 µl of HEPES-Tyrode's BSA buffer containing 0.5 mM IBMX for 15 min at 37 °C. The reaction was initiated by adding 100 µl of various concentrations of LPA in HEPES-Tyrode's BSA buffer. After 30 min of incubation at 37 °C, the reaction was terminated by adding 25 µl of lysis buffer. Cell lysates in a volume of 100 µl were used to determine the cAMP concentration using an enzyme immunoassay method.

Ca2+ Measurement—Cells serum-starved for 24 h were detached with PBS containing 2 mM EDTA, washed with HEPES-Tyrode's buffer, and then loaded with 3 µM Fura-2 AM (Dojindo, Kumamoto, Japan) in HEPES-Tyrode's BSA buffer for 1 h at 37 °C. The cells were washed twice and resuspended in HEPES-Tyrode's BSA buffer at a density of 1 x 106 cells/ml. The cell suspension (0.5 ml) was applied to a CAF-100 spectrofluorometer (Jasco, Tokyo, Japan), and 5 µ l of 100 µM LPA in HEPES-Tyrode's BSA buffer was added. The intracellular Ca2+ concentration ([Ca2+]i) was measured as the ratio of emission fluorescence at 500 nm in response to excitation at 340 and 380 nm.

Cell Rounding Assay—Cells (1 x 104) were seeded in poly-D-lysine-coated 12-well plates (BD Biosciences). After 24 h of incubation, the cells were washed three times with DMEM containing 0.1% BSA and serum-starved for 24 h. Three hours after a medium change, the cells were treated with 1 µM LPA for 15 min. The cells were examined for a round cell morphology lacking any neurite extensions or filopodia. Extended neurites were defined as having a length greater than the cell body. The number of rounded cells was expressed as a percentage of the observed cells (>200 cells/well).

Rho Inhibition Study—Cells (5 x 105) were seeded in poly-L-lysine-coated 35-mm dishes (Iwaki) in DMEM supplemented with 10% fetal bovine serum. After 24 h, either the Clostridium botulinum C3 exoenzyme expression vector (pEF-C3) (25) (a kind gift from Dr. S. Narumiya, Kyoto University, Kyoto, Japan) or the corresponding control vector (pEF-BOS) (26) (a kind gift from Dr. S. Nagata, Osaka University, Osaka, Japan) was cotransfected with an enhanced green fluorescent protein (EGFP) expression vector (pEGFP-C1; Clontech) at a 4:1 weight ratio, with 3 µg of total DNA, using the Lipofectamine 2000 reagent (Invitrogen). After 24 h, the cells were seeded in poly-D-lysine-coated 12-well plates and cultured for 24 h. The cells were then serum-starved for 12 h and treated with 1 µM LPA for 15 min. Following fixation with 1% paraformaldehyde for 15 min at 4 °C, EGFP images were obtained using a fluorescence microscope (Diaphoto, Nikon, Tokyo, Japan). EGFP-positive cells were examined for a round morphology without any neurite extensions or filopodia. At least 20 different fields were observed with a minimum of 100 EGFP-positive cells. The number of rounded cells was expressed as a percentage of the EGFP-positive cells.

Quantification of Cell Clustering—The degree of cell clustering was quantified by observing the distribution of the cell nuclei. Cells (1.5 x 105) were seeded in poly-D-lysine-coated 24-well plates (BD Biosciences). After 24 h of incubation, the cells were washed three times with DMEM containing 0.1% BSA and serum-starved for 24 h. Three hours after a medium change, the cells were treated with 1 µM LPA for 3 h, followed by fixation and staining with a Diff-Quik kit (Kokusai Shiyaku, Kobe, Japan). The distribution of the cells was mapped in rectangular areas (1710 x 1290 µm) by photographing the cultures (Cool Pix 990, Nikon). Each map was overlaid with grids at equal intervals (30 µm) and divided into 2451 unit squares. The randomness in spatial distribution was tested by counting the number of unit squares containing at least one nucleus. The intensity of the cell clustering was expressed as the percentage of the unit squares without any nuclei.

Cell Dissociation Assay—The Ca2+ sensitivity of cell-cell adhesion was estimated using trypsin treatment in the presence of either CaCl2 (TC treatment) or EDTA (TE treatment) as described (27, 28), with minor modifications. Briefly, 5 x 105 cells were seeded in poly-D-lysine-coated 35-mm dishes (BD Biosciences) and cultured overnight. After 24 h of serum starvation, the cells were stimulated with 1 µM LPA for 2 h and washed with HBSS containing either 2 mM CaCl2 or 2 mM EDTA. The washed cells were treated with 0.01% trypsin for 30 min at 37 °C and then dissociated by pipetting 10 times gently in 1 ml of HBSS with 0.01% trypsin. The number of cell clusters was counted with a particle counter (Beckman Coulter). The degree of cell-cell adhesion was expressed as the ratio of particles in the TC condition to particles in the TE condition (TC/TE). Negative control experiments without LPA treatment were also performed.

Western Blotting—Cells (4 x 106) were seeded in poly-L-lysine-coated 100-mm dishes. Following 24 h of serum starvation, the cells were treated with 1 µM LPA for 3 h, washed twice with PBS, and harvested in buffer B (25 mM HEPES-NaOH (pH 7.4), 10 mM MgCl2, and 0.25 M sucrose). The cells were centrifuged at 800 x g for 10 min at 4 °C, suspended in ice-cold buffer B containing 20 µM 4-amidinophenylmethylsulfonyl fluoride (Sigma) and a protease inhibitor mixture (Complete, Roche Applied Science), and sonicated three times for 30 s each at 4 °C. The cell debris was removed by centrifugation at 800 x g for 10 min at 4 °C. The protein concentration of the homogenate was determined with a Bradford assay (Bio-Rad) using BSA as a standard. Five micrograms of protein sample containing 5% 2-mercaptoethanol was analyzed by 7.5% SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was blocked with 5% skim milk (Difco) and probed with a mouse monoclonal antibody against N-cadherin or E-cadherin (BD Biosciences). The bands were visualized with an ECL chemiluminescence detection system (Amersham Biosciences) using horseradish peroxidaseconjugated anti-mouse IgG (Amersham Biosciences).

Immunofluorescence—Cells (3 x 105) were seeded into poly-L-lysine-coated glass-bottomed 35-mm dishes (Matsunami, Tokyo, Japan) and serum-starved for 24 h. Following stimulation with 1 µM LPA for 3 h at 37 °C, the cells were fixed with 4% paraformaldehyde for 20 min at 4 °C and rinsed twice with ice-cold PBS. Subsequently, the cells were incubated with a mouse monoclonal antibody against N-cadherin in PBS containing 1/4x permeabilization reagent (Beckman Coulter) for 1 h at room temperature. The primary antibody staining was visualized with an Alexa 488-conjugated goat anti-mouse IgG (Invitrogen). Images were obtained using an LSM510 laserscanning confocal microscope (Carl Zeiss, Jena, Germany) equipped with an argon laser as the light source.

Statistical Analysis—All values in the figures are expressed as means ± S.E. To determine statistical significance, the values were compared by analysis of variance followed by Tukey-Kramer test using Prism 4 software (GraphPad Software, San Diego, CA). The differences were considered significant if p values were less than 0.05.


Figure 1
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  		FIGURE 1.
Stable expression of LPA1 or LPA4 in B103 cells results in distinct morphologies. A, flow cytometry analysis. B103 cells were stably transfected with the expression vectors for LPA1 or LPA4, each tagged with an HA epitope at the N terminus. After staining with an anti-HA antibody and a phycoerythrin-conjugated secondary antibody, HA-positive cells were sorted with a cell sorter and then subcultured. Data shown are the surface expression levels of the HA epitope in subcultured polyclonal cells obtained by the second round of cell sorting. Empty vector-transfected polyclonal cells served as a negative control. B, morphology of B103-vector, B103-LPA1, and B103-LPA4 cells in serum-containing medium. The cells were photographed 24 h after seeding. Each stable cell line showed similar growth rate. Bar, 40 µm.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stable Expression of LPA1 and LPA4 in B103 Cells Results in Different Morphologies in Serum-containing Medium—To address the functional roles of LPA4 in neuronal cells, B103 rat neuroblastoma cells were stably transfected with the expression vector for either LPA1 or LPA4. B103 cells were selected because they lack endogenous responses to LPA (29, 30). Consistently, no specific binding was observed in B103-vector cells in the radioligand binding assays (see "Experimental Procedures"). To determine the intrinsic gene expression profiles of LPA receptors in B103 cells, we performed a reverse transcription-PCR analysis of total cellular RNA from the cells. Although LPA4 mRNA expression was slightly detected, no mRNA expression of the other three receptors, LPA1, LPA2, and LPA3, was observed (data not shown). This finding is consistent with a recent report by Tsukahara et al. (31). The apparent discrepancy between the expression of LPA4 and the lack of response to LPA might occur because the expression of LPA4 is too low to respond to LPA. Alternatively, post-transcriptional/translational modifications (32) may produce discordance between mRNA and protein expression. Thus, we discounted the low expression of LPA4 in B103 cells and took advantage of their unresponsiveness to LPA and their neuronal nature for the purpose of examining the functional roles of LPA4 in neuronal cells.

For the construction of stably transfected cell lines, LPA1 and LPA4 were tagged with an HA epitope at the N terminus to enable us to determine the levels of expression on the cell surfaces. Fluorescence-activated cell sorting enriched a polyclonal population of the drug-resistant cells that expressed each LPA receptor. These populations of stable clones are free of any clonal deviation that could cause functional variations. Following two rounds of cell sorting, we observed that the fluorescence intensity of B103-LPA1 cells was higher than that of B103-LPA4 cells (Fig. 1A), although the Bmax value for B103-LPA1 cells (0.8 pmol/mg of protein) was lower than that for B103-LPA4 cells (6.0 pmol/mg of protein). The apparent discrepancy might be because of two possibilities as follows: the usage of organellar membranerich microsome fractions and the difference in HA antibody immunoreactivity to the HA epitope tagged to two receptors. To confirm that no expression of the other subtypes of LPA receptors was enhanced secondary to the transfection, reverse transcription-PCR was performed with specific primers for LPA1, LPA2, LPA3, and LPA4 in B103-vector, B103-LPA1, and B103-LPA4 cells. As in the parental B103 cells, we observed only a low expression of LPA4 and virtually no expression of the other LPA receptors in all of the transfected cell lines (data not shown).

Although these stably transfected cell lines showed similar growth rates (data not shown), they showed distinctly different morphologies in serum-containing medium (Fig. 1B). As reported previously (33), B103-LPA1 cells displayed a flattened and more migratory morphology compared with B103-vector cells. Interestingly, B103-LPA4 cells had an epithelial like morphology and appeared to adhere more tightly to each other than B103-vector cells. These observations suggest that LPA1 and LPA4 have distinct signaling pathways that produce different cell morphologies.

LPA4 Does Not Affect Adenylyl Cyclase Activity in B103 Cells—We examined whether LPA4 mediates the inhibition of adenylyl cyclase activity in B103 cells, as the other three LPA receptors do (30) (Fig. 2A). In B103-LPA1 cells, LPA caused a dose-dependent inhibition of adenylyl cyclase activity with IC50 values below 10 nM (Fig. 2A). This inhibition was completely blocked by PTX treatment, indicating the primary role of Gi/o proteins. However, LPA at concentrations up to 10 µM did not blunt the forskolin-driven rises in cAMP accumulation in either B103-vector or B103-LPA4 cells, suggesting that LPA4 does not couple to Gi/o proteins.


Figure 2
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  		FIGURE 2.
LPA does not affect adenylyl cyclase activity in B103-LPA4 cells. A, failure of LPA to inhibit forskolin-induced cAMP accumulation in B103-LPA4 cells. Serum-starved B103-vector, B103-LPA1, and B103-LPA4 cells were stimulated with increasing concentrations of LPA in the presence of 0.5 mM IBMX and 50 µM forskolin. After a 30-min incubation at room temperature, the cells were solubilized, and cAMP concentrations in the cell lysates were measured. Forskolin-induced cAMP accumulation in the absence of LPA was set to 100%. Where indicated, the cells were pretreated with 100 ng/ml PTX for 24 h. Data are means ± S.E. (n = 4) of a representative of three independent experiments with similar results. B, failure of LPA to induce cAMP accumulation in B103-LPA4 cells. Serum-starved B103-vector, B103-LPA1, and B103-LPA4 cells were stimulated with increasing concentrations of LPA in the presence of 0.5 mM IBMX. After a 30-min incubation at room temperature, the cells were solubilized, and cAMP concentrations in the cell lysates were measured. The cells were pretreated with 100 ng/ml PTX for 24 h. Data are representative of three independent experiments with similar results.

 


Figure 3
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  		FIGURE 3.
LPA4-mediated Ca2+ mobilization is entirely dependent on Gq/11 proteins. B103-vector, B103-LPA1, and B103-LPA4 cells were serum-starved, loaded with 3 µM Fura-2 AM, and stimulated with 1 µM LPA or 10 µM ATP. Where indicated, the cells were pretreated with 5 µM YM-254890 for 10 min. Data are means ± S.E. (n = 3) of a representative of three independent experiments with similar results.

 
Previously, we reported that LPA induces cAMP accumulation in LPA4-expressing Chinese hamster ovary cells (19). However, LPA did not elevate basal cAMP levels in either B103-LPA4 cells or B103-LPA1 cells (Fig. 2B), suggesting that neither LPA1 nor LPA4 couples to Gs in B103 cells.

LPA1 and LPA4 Mediate Ca2+ Mobilization via Distinct Signaling Pathways—LPA has been shown to induce intracellular Ca2+ mobilization in many cell types (34), and all EDG family LPA receptor subtypes mediate Ca2+ mobilization when expressed in B103 cells (30). We therefore examined whether LPA4 mediates Ca2+ mobilization in B103 cells. Although B103-vector cells displayed no response to 1 µM LPA, increases in [Ca2+]i were observed both in B103-LPA1 and B103-LPA4 cells (Fig. 3). LPA induces phospholipase C-mediated Ca2+ duced Ca2+ response in B103-LPA4 cells and the ATP-induced Ca2+ response in both transfected cell lines were completely abolished by pretreatment with 5 µM YM-254890 (Fig. 3). In B103-LPA1 cells, YM-254890 only partially inhibited the LPA-induced response (Fig. 3), but the combination of PTX and YM-254890 produced complete inhibition (data not shown). The degree of inhibition with YM-254890 in B103-LPA1 cells was not altered at higher concentrations (up to 20 µM; data not shown), indicating that 5 µM YM-254890 was sufficient to inhibit the activation of Gq/11 proteins. These results suggest that both Gi/o and Gq/11 proteins mediate Ca2+ mobilization in B103-LPA1 cells, whereas Gq/11 is the dominant mediator of the response in B103-LPA4 cells.

Both LPA1 and LPA4 Mediate Cell Rounding via Rho-dependent and Gi/o- and Gq/11-independent Pathways—LPA induces rapid growth cone collapse, neurite retraction, and neuronal cell rounding in several neuronal cell types (10). Mouse LPA1 and LPA2 and human LPA1 have been reported to mediate LPA-induced cell rounding in B103 cells (29, 30, 33); we examined whether human LPA4 also mediates cell rounding in B103 cells by seeding cells at a low cell density (Fig. 4, A and B). Overexpression of LPA1 and LPA4 slightly increased the percentages of rounded cells even before LPA application. Within 15 min of LPA stimulation, about 80% of B103-LPA4 cells became rounded and underwent neurite retraction (Fig. 4A). Cell rounding was observed in B103-LPA1 cells as reported previously (33), but to a lesser degree than in B103-LPA4 cells. LPA-induced cell rounding was not observed in B103-vector cells.


Figure 4
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  		FIGURE 4.
LPA induces cell rounding in B103-LPA4 cells through a G12/13-Rho-ROCK-dependent pathway. A, induction of cell rounding in B103-LPA4 cells. Serum-starved B103-LPA4 cells were stimulated with 1 µM LPA for 15 min. Bar, 100 µm. B, effects of PTX, YM-254890, and Y-27632 on LPA-induced cell rounding in B103-vector, B103-LPA1, and B103-LPA4 cells. The cells were pretreated with either 100 ng/ml PTX for 24 h, 5 µM YM-254890 for 10 min, or 5 µM Y-27632 for 10 min. The percentages of rounded cells among >200 cells are shown. Data are means ± S.E. (n = 3) of a representative of three independent experiments with similar results. C, effects of C3 exoenzyme on LPA-induced cell rounding in B103-LPA4 cells. Either the C3 exoenzyme expression vector or the corresponding control vector was cotransfected with the EGFP expression vector. The cells were seeded, serum-starved, and treated with 1 µM LPA for 15 min. Following the fixation, EGFP images were obtained using a fluorescence microscope. The percentages of rounded cells among >100 EGFP-positive cells are shown. Data are means ± S.E. (n = 3) of a representative of two independent experiments with similar results.

 
The role of Rho in LPA-induced cell rounding is now well established (36), and the G12/13 types of heterotrimeric G proteins are known to be upstream activators of Rho proteins (1, 2, 7). On the other hand, there are reports that Gq/11 activation induces cell rounding through Rho-dependent (37) and -independent (38) pathways. To determine which G proteins and signaling molecules are involved in LPA-induced cell rounding, we pretreated the cells with PTX, YM-254890, and a ROCK inhibitor, Y-27632 (Fig. 4B). In B103-LPA4 cells, neither PTX nor YM-254890 inhibited LPA-induced cell rounding; in contrast, Y-27632 completely inhibited this morphological change. Y-27632 also hampered LPA-induced cell rounding in B103-LPA1 cells, whereas YM-254890 did not affect the number of rounded cells. Interestingly, pretreatment with PTX increased the degree of LPA-induced cell rounding in B103-LPA1 cells. To confirm the involvement of Rho, B103-LPA4 cells were transfected with C3 exoenzyme, which inactivates Rho by ADP-ribosylation. The transfected cells were identified by cotransfection of an EGFP expression construct. C3 exoenzyme transfection blunted LPA-induced cell rounding in B103-LPA4 cells, again indicating the involvement of Rho (Fig. 4C).

LPA4 Mediates ROCK-dependent Cell Aggregation—As described earlier, B103-LPA4 cells appeared to form aggregates in serum-containing medium to a greater extent than B103-vector cells (Fig. 1B). To determine whether the binding of LPA to LPA4 mediates the induction of cell-cell adhesion, B103-LPA4 cells at a medium cell density were stimulated with 1 µM LPA after 24 h of serum starvation. Although the rapid cell rounding after LPA application was difficult to evaluate at this cell density because of the formation of cell-cell contacts, LPA caused a slow but dramatic aggregation in B103-LPA4 cells (Fig. 5A, panel f). The morphological change observed in B103-LPA4 cells was transient, reaching a maximum 2-3 h after the treatment and then returning to the base line 24 h after the treatment (data not shown).

To investigate the signaling pathways downstream of LPA4 that are involved in the cell aggregation, we treated B103-LPA4 cells with several inhibitors. The LPA-induced morphological changes in B103-LPA4 cells were completely prevented by Y-27632 (Fig. 5A, panel o). In contrast, neither PTX nor YM-254890 inhibited the cell aggregation (Fig. 5A, panels i and l). We quantified the degree of cell aggregation by examining the randomness in the spatial distribution of the cells (see the "Experimental Procedures"; Fig. 5B). These results suggest that Rho mediates LPA-induced cell aggregation in B103-LPA4 cells inaGi/o- and Gq/11-independent manner. Rho regulates the reorganization of the actin cytoskeleton, which can modify the intensity of adhesion (28, 39). To examine whether the reorganization of the actin cytoskeleton was involved in this effect, B103-LPA4 cells were pretreated with cytochalasin D (an inhibitor of actin polymerization). In these cells, morphological changes were not observed after LPA stimulation, indicating that actin reorganization is involved in the LPA-induced cell aggregation (data not shown). Like B103-LPA4 cells, PTX-treated B103-LPA1 cells became aggregated after LPA stimulation (Fig. 5A, panel h).


Figure 5
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  		FIGURE 5.
Cell aggregation in B103-LPA4 cells is mediated by ROCK. A, cells were pretreated with either 100 ng/ml PTX for 24 h, 5 µM YM-254890 for 10 min, or 5 µM Y-27632 for 10 min prior to the LPA stimulation for 3 h. Bar, 100 µm. B, quantification of cell clustering. Serum-starved cells were treated with 1 µM LPA for 3 h, followed by fixation and staining. The intensity of the cell clustering was calculated as described under the "Experimental Procedures." Data are means ± S.E. of three different rectangular areas (one rectangular area/well) of two independent experiments with similar results.

 
LPA4 Mediates N-cadherin-dependent Cell-Cell Adherence—Through LPA-induced cell aggregation, B103-LPA4 cells formed tightly compact aggregates (Fig. 5A, panel f), which dissociated very little after pipetting (data not shown). Cell-cell adhesion mechanisms can be Ca2+-dependent and Ca2+-independent, and cadherins are the major components of the Ca2+-dependent system. Cadherin-dependent adhesion was originally defined as being trypsin-resistant in the presence of Ca2+ and trypsin-sensitive in the absence of Ca2+ (40). We examined whether LPA-induced cell adhesion was mediated by cadherins using a cell dissociation assay, one of the adhesion assays for the evaluation of the cadherin activity (27, 28). We defined the TC/TE index as a ratio of the cell particle number after trypsin treatment in the presence of Ca2+ (TC) to the number after trypsin treatment with EDTA (TE). Cadherin-dependent adhesion remains after trypsin-Ca2+ treatment, whereas trypsin-EDTA treatment disrupts cell adhesion nearly completely. In either treatment, an increase in particles would occur when a large aggregate breaks into small particles by pipetting; the higher the number of particles, the lower the aggregation (adhesion). Thus, the TC/TE index is negatively correlated with cadherin-mediated adhesion. The aggregation level of B103-LPA4 cells increased after LPA stimulation (Fig. 6A; note that the index inversely reflects cadherin activity). LPA treatment did not significantly affect the TC/TE index in either B103-vector cells or B103-LPA1 cells. These results suggest that LPA increased the cadherin-mediated adhesive activity in B103-LPA4 cells. Even LPA-untreated B103-LPA4 cells had significantly more cadherin-dependent adhesion activity, i.e. a lower TC/TE index, than LPA-untreated B103-vector and B103-LPA1 cells (Fig. 6A).

The cadherins constitute a large superfamily of molecules that includes the classic cadherins, the desmosomal cadherins, the protocadherins, and the cadherin-like signaling receptors (41). The levels of the two classic cadherins most commonly expressed in the nervous system, N- and E-cadherin, were determined by Western blotting of LPA-treated or -untreated B103 cells. Consistent with a previous report (42), these cells abundantly expressed N-cadherin (Fig. 6B), whereas E-cadherin was undetectable (data not shown). The expression level of N-cadherin was not up-regulated by LPA treatment in any of the transfected cell lines (Fig. 6B), and N-cadherin was intact in the cells undergoing TC treatment. In contrast, TE treatment resulted in complete digestion of N-cadherin (data not shown), as reported previously (43). We next examined whether LPA increases N-cadherin-mediated cell-cell contacts. LPA promoted the assembly of N-cadherin in the form of a thick, bright band at the cell-cell contact area in B103-LPA4 cells but not in B103-vector cells (Fig. 6C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A number of studies have shown that LPA mediates morphological changes in neuronal cells through the Rho-ROCK pathway (10, 11, 44). It has been proposed that these effects are mediated by LPA1 and/or LPA2 (10, 11, 44). Recently, we identified p2y9/GPR23 as a fourth LPA receptor (LPA4) that is structurally distinct from the EDG family of LPA receptors (19). The expression of LPA4 in neuronal cells implies a significant role for this receptor in the nervous system (18). The results in this study demonstrate that LPA4 caused morphological changes in B103 neuronal cells, including cell rounding and N-cadherin-associated cell aggregation, both of which were mediated by the Rho-ROCK pathway.


Figure 6
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  		FIGURE 6.
LPA induces N-cadherin-dependent cell adhesion in B103-LPA4 cells. A, Ca2+ dependence of cell-cell adhesion in B103-LPA4 cells. Serum-starved cells were stimulated with 1 µM LPA for 2 h and washed with HBSS containing either 1 mM Ca2+ or 1 mM EDTA. The washed cells were treated with 0.01% trypsin for 30 min in the presence of either Ca2+ (TC treatment) or EDTA (TE treatment) at 37 °C. Then the cells were dissociated by pipetting 10 times, and the number of particles was counted with a particle counter. The degree of cell-cell adhesion was expressed as the ratio of TC/TE. Note that the ratio inversely reflects cadherin activity. Negative control experiments without LPA treatment were also performed. Data are means ± S.E. of five independent experiments. *, p < 0.05; **, p < 0.001 (using analysis of variance followed by Tukey-Kramer test). B, expression of N-cadherin protein in B103 cells. Serum-starved cells were incubated with or without 1 µM LPA for 3 h and lysed. The same amount of protein was subjected to Western blot (WB) analysis. C, immunostaining of N-cadherin in B103-vector and B103-LPA4 cells. Serumstarved cells were stimulated with 1 µM LPA, stained with an antibody against N-cadherin, and visualized with a fluorescein-labeled secondary antibody. Negative control experiments without LPA treatment were also performed. Bar, 100 µm.

 
Ca2+ mobilization and adenylyl cyclase inhibition are the major cellular responses to LPA (45). When expressed in B103 neuronal cells, each of the EDG family LPA receptors, LPA1, LPA2, and LPA3, mediates both of these reactions (30). LPA1 is likely to mediate the Ca2+ response through Gq/11 proteins (Fig. 3). The PTX-sensitive inhibition of adenylyl cyclase (Fig. 2A) suggests that LPA1 also couples to Gi/o. We showed that LPA4 mediates the Ca2+ response in B103 cells (Fig. 3). This is consistent with our previous report (19) that the stable expression of LPA4 in Chinese hamster ovary cells significantly enhanced the LPA-induced Ca2+ mobilization. From our results using YM-254890, LPA4 probably mediates Ca2+ mobilization through Gq/11 proteins (Fig. 3). In contrast, LPA did not inhibit adenylyl cyclase in B103-LPA4 cells (Fig. 2A). These results indicate that unlike the other LPA receptor subtypes, LPA4 does not couple to Gi/o proteins.

Neurite retraction and neurite formation play a role in the remodeling of neurons for guidance and synaptic plasticity (46). Neurite retraction in neuronal cells is induced by lysophospholipids, including LPA and sphingosine 1-phosphate (S1P), in addition to semaphorins, netrins, and ephrins (10, 11, 47). LPA induces neurite retraction through LPA1 or LPA2 when expressed in B103 cells (30). In this study, we showed that 1 µM LPA induced cell rounding in B103-LPA4 cells (Fig. 4A). Sugiura et al. (48) reported that rat brain contains 3.73 nmol of LPA/g of tissue. These results suggest a role for LPA4 in LPA-induced neurite retraction. Neurite initiation and formation involve actin cytoskeletal changes, and as a regulator of actin reorganization, Rho GTPase has a profound effect on neuritogenesis (47). For example, S1P induces Rho-dependent neurite retraction through the S1P2 (49, 50), S1P3 (49), and S1P5 receptors (50, 51). Several studies have also revealed a critical role for Rho and ROCK in LPA-induced neurite retraction (36, 52, 53), although some studies have reported Rhoindependent neurite retraction (38, 54). Judging from its complete inhibition by C3 exoenzyme and Y-27632, the cell rounding induced by LPA4 depended on Rho and ROCK in B103-LPA4 cells (Fig. 4, B and C).

In general, activation of G12/13 proteins leads to an increase in RhoA guanine nucleotide exchange, activation of ROCK, and actin polymerization (55, 56), although some studies have implied that Gq/11 proteins can also activate Rho (57, 58). Based on our results, it is conceivable that the G12/13 proteins are upstream regulators of Rho in B103-LPA1 cells and B103-LPA4 cells, because YM-254890 inhibited cell rounding in both cell types (Fig. 4B). In many studies reporting the induction of neurite retraction by LPA (10), the LPA receptor subtypes responsible have not been identified, or LPA1 and LPA2 were suggested as candidate subtypes based on mRNA expression in the neuronal cells examined (59-62). This study suggests the possibility that LPA4 was involved in the neurite retraction in some of these studies.

LPA has been shown to stimulate cell motility and to modulate tumor cell invasion, both of which are mediated mainly by LPA1 and Gi/o proteins (33, 63, 64). In the presence of serum, B103-LPA1 cells exhibited a flattened morphology and were widely dispersed throughout the dish (Fig. 1B) (33). This morphological phenotype was probably evoked by LPA through the LPA1-Gi/o-Rac signaling axis (33). In sharp contrast to B103-LPA1 cells, B103-LPA4 cells formed cell aggregates in serum-containing medium (Fig. 1B), apparently through activation of the G12/13-Rho-ROCK signaling axis (Fig. 5). This cell-cell adhesion involved N-cadherin without de novo synthesis (Fig. 6). Because Rho affects cadherin-dependent adhesion through actin cytoskeleton reorganization (28), we presume that LPA-induced cytoskeletal changes affect the subcellular distribution of N-cadherin, as shown in Fig. 6C, leading to strong cell-cell adhesion in B103-LPA4 cells. This is supported by the current results that treatment with Y-27632 and cytochalasin D abolished the LPA-induced cell aggregation (Fig. 5 and data not shown). N-cadherin is widely expressed in the nervous system and has critical roles in neural development and functions, including synapse formation and myelination. Weiner et al. (65) previously reported that LPA induced cell-cell junctions containing N-cadherin in rat Schwann cells. Furthermore, LPA was reported to induce cell clustering in neural progenitor cells prepared from embryonic rat hippocampus (66) and in mouse postmitotic cortical neurons (16). Our results raise the possibility that in addition to LPA1 and LPA2, LPA4 might also be involved in these effects in neural cells and have critical roles in the development and function of the nervous system. In contrast to LPA1 and LPA2, which activate Rac through Gi/o (17, 64), LPA4 is unlikely to activate the Gi/o-Rac pathway because LPA4 did not inhibit adenylyl cyclase activity (Fig. 3A). Therefore, LPA4 might have a unique role in keeping a proper balance between Rho and Rac activation, which is important for neuronal development and function (47).

We observed that PTX significantly enhanced the intensity of LPA1-mediated cell rounding (Fig. 4B). This "permissive effect" is consistent with a previous report that PTX enables LPA-induced cell rounding in 1321N1 astrocytoma cells (61). It is known that Rho activity is inhibited by Rac activation through Gi/o proteins (67). Indeed, Rac activation functionally antagonizes Rho-mediated neurite retraction in 1321N1 astrocytoma cells (61). LPA1 was shown to couple to Gi/o and activate Rac strongly in B103 cells (33) and other cells, including mouse embryonic meningeal fibroblast (MEMF) and mouse skin fibroblast (MSF) cells (17, 64). Taken together, we suggest that PTX treatment of B103-LPA1 cells suppresses Gi/o proteins and subsequently suppresses Rac activation by LPA, which in turn permits Rho-mediated cell rounding. This mechanism probably also accounts for the LPA-induced aggregation of PTX-treated B103-LPA1 cells (Fig. 5, A, panel h, and B).

We showed here that LPA4 has Rho-dependent morphological effects. It has been reported that LPA-induced Rho activation is mediated by LPA1 and/or LPA2. However, pathways independent of LPA1 and LPA2 have also been proposed. Contos et al. (17) showed that MEMF cells from LPA1 and LPA2 double knockouts remained capable of forming stress fibers in response to LPA. This study proposed the presence of unknown LPA receptors in MEMF cells because of the absence of LPA3 mRNA. Consistent with this, Hama et al. (64) reported that LPA activates Rho in MSF cells from LPA1 and LPA2 double knockouts. Our results, together with the abundant expression of LPA4 in MSF cells (64), suggest that LPA4 may also be involved in LPA-induced Rho activation in these cells. Furthermore, Hama et al. (64) observed that Rac activation was totally dependent on LPA1 and LPA2, supporting our hypothesis that LPA4 does not activate the Gi/o-Rac signaling axis.


Figure 7
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  		FIGURE 7.
LPA1 and LPA4 have distinct signaling pathways that produce different cell morphologies in B103 cells. LPA4 expression results in cell rounding and aggregated morphology through G12/13-Rho-ROCK pathway. In contrast, LPA1 expression results in flattened and dispersed morphology as reported previously (33). Inactivation of Gi/o proteins in LPA1-expressing cells by PTX treatment leads to "LPA4-expressing cells like" aggregated morphology, suggesting the involvement of the inhibitory effect of Gi/o on Rho activation probably through Rac. Both LPA1 and LPA4 couple to Gq, although Gq-involved pathway does not affect cell morphology.

 
We also observed that the stable expression of LPA1 or LPA4 slightly increased the population of rounded cells even before LPA application (Fig. 4B). Furthermore, serum-starved B103-LPA4 cells adhered to one another more strongly than B103-vector or B103-LPA1 cells did (Fig. 6A). These morphological effects might be because of the constitutive activation of these LPA receptors. Indeed, there are many reports showing that the constitutive expression of GPCR for lipid mediators, including lysophospholipids and prostanoids, has morphological effects (30, 49, 68, 69). For example, cell rounding and cadherin-dependent adhesion in the absence of ligand occur in human embryonic kidney 293 cells transfected with FPB receptor, an isoform of prostanoid GPCR (69, 70). The involvement of phosphatidylinositol 3-kinase and beta-catenin was proposed for these constitutive activities (70). Another conceivable explanation for the morphological effects observed in serum-starved B103-LPA1 and B103-LPA4 cells involves autocrine ligand secretion and subsequent receptor activation (33). Both hypotheses are consistent with our results that the treatment of these cells with Y-27632 decreased the percentage of rounded cells (Fig. 4B).

In summary, as shown in Fig. 7, we demonstrate for the first time that the novel LPA receptor subtype LPA4 is coupled to the activation of Rho in a rat neuronal cell line. The activation of Rho through LPA4 leads to morphological changes, including cell rounding and cell aggregation. LPA is well known to induce neurite retraction and cell clustering in neural cells. The identification of Rho as an effector of LPA4 will give insight into some of the physiological and morphological effects of LPA that could not be explained by the EDG family LPA receptors. A full understanding of the potential roles of the endogenous LPA4 receptor in the development and function of the nervous system awaits future studies.


   FOOTNOTES
 
* This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. S. and S. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: mame{at}m.u-tokyo.ac.jp.

2 The abbreviations used are: LPA, lysophosphatidic acid; GPCR, G protein-coupled receptor; EDG, endothelial differentiation gene; ROCK, Rho-associated kinase; DMEM, Dulbecco's modified Eagle's medium; PTX, pertussis toxin; HA, hemagglutinin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HBSS, Hank's balanced salt solution; IBMX, 2-isobutyl-1-methylxanthine; [Ca2+]i, intracellular Ca2+ concentration; EGFP, enhanced green fluorescence proteins; S1P, sphingosine 1-phosphate; MEMF, mouse embryonic meningeal fibroblast; MSF, mouse skin fibroblast. Back


   ACKNOWLEDGMENTS
 
We thank Drs. T. Yokomizo and T. Okuno (Kyushu University) for vital discussions and critical suggestions. We also thank Dr. J. Chun (The Scripps Research Institute, La Jolla, CA) for providing B103 rat neuroblastoma cells; Dr. J. Miyazaki (Osaka University) for pCXN2; Drs. S. Narumiya (Kyoto University) and S. Nagata (Osaka University) for the expression vector for C3 exoenzyme (pEF-C3) and its parental vector (pEF-BOS), respectively; and Dr. J. Takasaki (Astellas Pharma, Tokyo, Japan) for YM-254890.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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