guanosine-triphosphate and sphingosine-1-phosphate

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structure-activity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P(1) and S1P(3) receptors. The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.

Topics: Calcium; Cell Line; Cell Membrane; Cell Movement; Dose-Response Relationship, Drug; Drug Design; Fingolimod Hydrochloride; Guanosine Triphosphate; Humans; Inhibitory Concentration 50; Kinetics; Ligands; Lipids; Lymphocytes; Lysophospholipids; Models, Chemical; Phosphoserine; Propylene Glycols; Protein Binding; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Structure-Activity Relationship; Transfection

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.

Topics: Adenoviridae; Animals; Blotting, Northern; Blotting, Western; Calcium; Cell Movement; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genetic Vectors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Ligands; Lysophospholipids; Melanoma, Experimental; Mice; Neoplasm Invasiveness; Phosphorylcholine; Plasmids; Protein Isoforms; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Temperature; Time Factors

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha(13). Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p115 RhoGEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252--288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p115 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sphingosine 1-phosphate was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.

Topics: 3T3 Cells; Animals; Baculoviridae; Binding Sites; Cell Line; Cell Membrane; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Lysophospholipids; Mice; Recombinant Fusion Proteins; Recombinant Proteins; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; Sequence Deletion; Sphingosine; Spodoptera; src Homology Domains; Transfection

1. The Ca2+ entry pathway activated by sphingosine-1-phosphate (S1P) was examined in primary cultured vascular endothelial cells dispersed from human umbilical vein (HUVECs) by measuring intracellular Ca2+ concentration ([Ca2+]i), whole-cell membrane currents and single channel activity. 2. Application of S1P to HUVECs induced a slowly developing, sustained increase in [Ca2+]i. When Ca2+ was absent from the bathing solution, no S1P-induced changes in [Ca2+]i were observed. Tert-butylhydroquinone (BHQ), an inhibitor of Ca2+ pumps in endoplasmic reticulum, and histamine induced a transient elevation of [Ca2+]i in HUVECs. 3. Pretreatment of HUVECs with 100 ng x ml(-1) pertussis toxin (PTX) for 15 h almost abolished the S1P effect on [Ca2+]i and reduced the histamine effect to 40% of the control. The BHQ-induced elevation of [Ca2+]i was insensitive to PTX. 4. When whole-cell membrane currents were recorded using the amphotericin B-perforated-patch clamp technique while monitoring [Ca2+]i, application of S1P induced a tiny inward current (I(S1P)) which was followed by the elevation of [Ca2+]i. I(S1P) reversed at +20.0 +/- 2.7 mV under these experimental conditions. 5. When S1P was included in the pipette solution in the excised inside-out patch clamp configuration, single channel activity with a conductance of 17 pS was activated. This channel activity depended on the presence of intracellular GTP. 6. In summary, these results show that S1P has a novel effect in mammalian cardiovascular endothelium to activate a non-selective cation (NSC) channel in a GTP-dependent manner via a PTX-sensitive G-protein. This S1P-sensitive NSC channel acts as a Ca2+ entry pathway in endothelium.

Topics: Calcium; Cations; Cells, Cultured; Electric Conductivity; Endothelium, Vascular; Guanosine Triphosphate; Humans; Intracellular Membranes; Ion Channels; Lysophospholipids; Patch-Clamp Techniques; Sphingosine