3-amino-4-(3-hexylphenylamino)-4-oxobutylphosphonic-acid and sphingosine-1-phosphate

We conducted this experimental study to analyze the relationship between sphingosine-1-phosphate (S1P)-induced mitogen-activated protein (MAP) kinase pathways and keloid formation. We collected samples of the normal tissue and the keloid tissue from 10 normal healthy individuals and 12 patients with keloid scars, respectively. Then, we compared the level of sphingosine-1-phosphate receptor (S1PR1/S1PR2) mRNA/protein expression between the normal tissue and the keloid tissue. Moreover, we also compared the level of S1PR protein expression, that of S1P-induced COL1A1 (collagen Type I, α-1 chain) expression, that of S1P-induced JNK/ERK phosphorylation, that of S1P-induced COL1A1 expression following the treatment with 30 μM PD98059 (ERK inhibitor) or 30 μM SP600125 (JNK inhibitor) and that of S1P-induced COL1A1 expression following the treatment with W146 (S1PR1 inhibitor) or JTE013 (S1PR2 inhibitor) between the normal fibroblasts and the keloid fibroblasts. We found that the level of S1PR1/S1PR2 mRNA/protein expression was significantly higher in the keloid tissue as compared with the normal tissue. Our results also showed that the level of S1P-induced COL1A1 expression and that of S1P-induced JNK/ERK phosphorylation were significantly higher in the keloid fibroblasts as compared with the normal ones (P < 0.05). Furthermore, there were significant decreases in the level of S1P-induced COL1A1 expression when the keloid fibroblasts were treated with 30 μM SP600125 or 30 μM PD98059 and that of S1P-induced COL1A1 expression when the treated with 100 nM W146 or 100 nM JTE013 (P < 0.05). Our results indicate that S1P-induced signal transduction is associated with increased collagen synthesis via S1PR-mediated signaling pathways in the keloid tissue.

Topics: Adult; Anilides; Anthracenes; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Fibroblasts; Flavonoids; Humans; Keloid; Lysophospholipids; Male; MAP Kinase Signaling System; Middle Aged; Mitogen-Activated Protein Kinases; Organophosphonates; Phosphorylation; Pyrazoles; Pyridines; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Young Adult

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration.. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied.. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect.. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies.

Topics: Anilides; Aspirin; Blood Platelets; Cell Movement; Cells, Cultured; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Organophosphonates; Receptors, Lysosphingolipid; Sphingosine

Overexpression of P-glycoprotein (P-gp) in the brain is an important factor leading to drug-resistant epilepsy. Clinical use of P-gp inhibitors is limited by their systemic toxicity. In this study, we tested the hypothesis that FTY720, a sphingosine 1-phosphate (S1P) analogue used for treating multiple sclerosis, modulates the up-regulation of P-gp and improves brain delivery of phenytoin (PHT) through S1P receptor 1 in the hippocampus of a pilocarpine-induced rat model of status epilepticus (SE). We administered vehicle, FTY720 or FTY720+ W146 (an S1P receptor 1 antagonist) to SE rats. Forty-eight hours after SE, we dissected the hippocampus and measured P-gp expression, NF-κB activity and levels of inflammatory mediators (TNF-α and COX-2) by Western blotting and enzyme-linked immunosorbent assay. We also measured hippocampal and plasma concentrations of PHT 30, 60, 90, 120 and 180 min. after an intraperitoneal injection of PHT (50 mg/kg) 48 hr after SE, using microdialysis and high-performance liquid chromatography. FTY720 alleviated the overexpression of hippocampal P-gp in SE rats and reduced NF-κB activity and TNF-α and COX-2 expression, and W146 blocked the effects of FTY720. Furthermore, SE rats that received FTY720 showed significantly greater hippocampal extracellular PHT concentrations than those that received vehicle, and W146 abolished this effect. Our results suggest that FTY720 alleviates seizure-induced overexpression of P-gp by inhibiting S1P receptor 1-mediated inflammation in rat hippocampus and improves PHT delivery to brain. FTY720 shows potential as an adjuvant therapy for drug-resistant epilepsy.

Topics: Anilides; Animals; Anticonvulsants; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclooxygenase 2; Disease Models, Animal; Drug Resistant Epilepsy; Fingolimod Hydrochloride; Hippocampus; Humans; Immunosuppressive Agents; Injections, Intraperitoneal; Lysophospholipids; Male; Organophosphonates; Phenytoin; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Up-Regulation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000-10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts.

Topics: Anilides; Cell Line, Tumor; Chemotaxis; Gene Expression Regulation, Leukemic; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Organophosphonates; Phosphorylation; Proto-Oncogene Proteins c-akt; rac1 GTP-Binding Protein; Receptors, Lysosphingolipid; RNA, Messenger; Signal Transduction; Sphingosine; T-Lymphocytes

FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.

Topics: Analgesics; Anilides; Animals; In Situ Hybridization; Lysophospholipids; Male; Mice; Neuralgia; Organophosphates; Organophosphonates; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Wounds and Injuries

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1-5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability.. Isolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs).. After siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons.. These results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3.

Topics: Action Potentials; Anilides; Animals; Cells, Cultured; Dinoprostone; Enzyme Inhibitors; Fingolimod Hydrochloride; Ganglia, Spinal; Gene Expression Regulation; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, Inbred C57BL; Organophosphonates; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA, Small Interfering; Sensory Receptor Cells; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiazolidines

Sphingosine-1-phosphate (S1P) is a ceramide derivative serving not only as a regulator of immune properties but also as a modulator of brain functions. To better understand the mechanism underlying the effects of S1P on brain functions, we investigated the potential impact of S1P receptor (S1PR) activation on NMDA receptor subunits. We used acute rat hippocampal slices as a model system, and determined the effects of the active phosphorylated S1P analog, fingolimod (FTY720P) on various NMDA receptors. Treatment with FTY720P significantly increased phosphorylation of GluN2B-containing NMDA receptors at Tyr1472. This effect appears rather specific, as treatment with FTY720P did not modify GluN2B-Tyr1336, GluN2B-Ser1480, GluN2A-Tyr1325 or GluN1-Ser897 phosphorylation. Pre-treatment of hippocampal slices with the compounds W146 and PP1 indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 epitopes was dependent on activation of S1PR subunit 1 (S1PR1) and Src/Fyn kinase, respectively. Cell surface biotinylation experiments indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 was also associated with increased levels of GluN1 and GluN2B subunits on membrane surface, whereas no change was observed for GluN2A subunits. We finally demonstrate that FTY720P is inclined to favor Tau and Fyn accumulation on plasma membranes. These results suggest that activation of S1PR1 by FTY720P enhances GluN2B receptor phosphorylation in rat hippocampal slices, resulting in increased levels of GluN1 and GluN2B receptor subunits in neuronal membranes through a mechanism probably involving Fyn and Tau.

Topics: Analysis of Variance; Anilides; Animals; Biotinylation; Cell Membrane; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Lysophospholipids; Male; Organophosphates; Organophosphonates; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Receptors, N-Methyl-D-Aspartate; Sphingosine; Sphingosine-1-Phosphate Receptors; src-Family Kinases; Time Factors; Transcriptional Activation; Up-Regulation

The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy- induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents.We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR(1))- dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/ neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration- approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.

Topics: Anilides; Animals; Antineoplastic Agents, Phytogenic; Cytokines; Enzyme Activation; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Indans; Lysophospholipids; Male; Neuralgia; Organophosphonates; Oxadiazoles; Paclitaxel; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiazoles; Thiophenes

In addition to an extensively characterized role of high-density lipoprotein (HDL) in reverse cholesterol transport, bioactive lipids bound to HDL can also exert diverse vascular effects. Despite this, integration of HDL action in the vasculature with pathways that metabolize HDL and release bioactive lipids has been much less explored. The effects of HDL on endothelial cells are mediated in part by HDL-associated sphingosine 1-phosphate (S1P), which binds to S1P1 receptors and promotes activation of endothelial NO synthase (eNOS) and the kinase Akt. In these studies, we characterized the role of endothelial lipase (EL) in the control of endothelial signaling and biology, including those mediated by HDL-associated S1P.. HDL-induced angiogenesis in aortic rings from EL-deficient (EL(-/-)) mice was markedly decreased compared with wild-type controls. In cultured endothelial cells, small interfering RNA-mediated knockdown of EL abrogated HDL-promoted endothelial cell migration and tube formation. Small interfering RNA-mediated EL knockdown also attenuated HDL-induced phosphorylation of eNOS(1179) and Akt(473). S1P stimulation restored HDL-induced endothelial migration and Akt/eNOS phosphorylation that had been blocked by small interfering RNA-mediated EL knockdown. HDL-induced endothelial cell migration and Akt/eNOS phosphorylation were completely inhibited by the S1P1 antagonist W146 but not by the S1P3 antagonist CAY10444.. EL is a critical determinant of the effects of HDL on S1P-mediated vascular responses and acts on HDL to promote activation of S1P1, leading to Akt/eNOS phosphorylation and subsequent endothelial migration and angiogenesis. The role of EL in HDL-associated S1P effects provides new insights into EL action, the responses seen through EL and HDL interaction, and S1P signaling.

Topics: Anilides; Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Cholesterol; Endothelium, Vascular; Lipase; Lipoproteins, HDL; Lysophospholipids; Mice; Mice, Knockout; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Organophosphonates; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine

The sphingosine 1-phosphate receptor 1 (S1P₁) and its ligand, sphingosine 1-phosphate (S1P), have now emerged as critical regulators of lymphocyte trafficking, vascular development and integrity, and immunity. S1P₁ is targeted by the phosphorylation product of fingolimod, which has been approved for the treatment of multiple sclerosis. The recent progress in the structural biology of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors has now enabled the elucidation of the structure of S1P₁. Analysis of the structure, along with structure activity and mutagenesis analysis, highlighted key interactions associated with the binding of S1P and agonists and suggested that the ligand may gain access to the binding pocket by lateral diffusion within the plasma membrane. The S1P₁ crystal structure will be helpful for designing ligands that specifically target S1P₁.

Topics: Anilides; Binding Sites; Crystallography, X-Ray; Drug Design; Humans; Indans; Lysophospholipids; Models, Molecular; Organophosphonates; Oxadiazoles; Protein Binding; Protein Conformation; Receptors, Lysosphingolipid; Sphingosine

Previously we demonstrated that sphingosine 1-phosphate receptor 1 (S1PR(1)) played a prominent, but not exclusive, role in enhancing the excitability of small-diameter sensory neurons, suggesting that other S1PRs can modulate neuronal excitability. To examine the potential role of S1PR(2) in regulating neuronal excitability we used the established selective antagonist of S1PR(2), JTE-013. Here we report that exposure to JTE-013 alone produced a significant increase in excitability in a time- and concentration-dependent manner in 70-80% of recorded neurons. Internal perfusion of sensory neurons with guanosine 5'-O-(2-thiodiphosphate) (GDP-β-S) via the recording pipette inhibited the sensitization produced by JTE-013 as well as prostaglandin E(2). Pretreatment with pertussis toxin or the selective S1PR(1) antagonist W146 blocked the sensitization produced by JTE-013. These results indicate that JTE-013 might act as an agonist at other G protein-coupled receptors. In neurons that were sensitized by JTE-013, single-cell RT-PCR studies demonstrated that these neurons did not express the mRNA for S1PR(2). In behavioral studies, injection of JTE-013 into the rat's hindpaw produced a significant increase in the mechanical sensitivity in the ipsilateral, but not contralateral, paw. Injection of JTE-013 did not affect the withdrawal latency to thermal stimulation. Thus JTE-013 augments neuronal excitability independently of S1PR(2) by unknown mechanisms that may involve activation of other G protein-coupled receptors such as S1PR(1). Clearly, further studies are warranted to establish the causal nature of this increased sensitivity, and future studies of neuronal function using JTE-013 should be interpreted with caution.

Topics: Action Potentials; Analysis of Variance; Anilides; Animals; Capsaicin; Cell Line, Tumor; Cell Movement; Dinoprostone; Dose-Response Relationship, Drug; Drug Interactions; Ganglia, Spinal; Guanosine Diphosphate; Hyperalgesia; Lysophospholipids; Male; Melanoma; Mice; Organophosphonates; Pain Threshold; Patch-Clamp Techniques; Pertussis Toxin; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sensory Receptor Cells; Sensory System Agents; Sphingosine; Thionucleotides; Time Factors; Wound Healing

Sphingosine-1-phosphate (S1P) is an important mediator of inflammation recently shown in in vitro studies to increase the excitability of small-diameter sensory neurons, at least in part, via activation of the S1P(1) receptor subtype. Activation of S1PR(1) has been reported to increase the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide (O(2)(·-)) and nitric oxide synthase (NOS)-derived nitric oxide (NO). This process favors the formation of peroxynitrite (ONOO(-) [PN]), a potent mediator of hyperalgesia associated with peripheral and central sensitization. The aims of our study were to determine whether S1P causes peripheral sensitization and thermal hyperalgesia via S1PR(1) activation and PN formation. Intraplantar injection of S1P in rats led to a time-dependent development of thermal hyperalgesia that was blocked by the S1PR(1) antagonist W146, but not its inactive enantiomer W140. The hyperalgesic effects of S1P were mimicked by intraplantar injection of the well-characterized S1PR(1) agonist SEW2871. The development of S1P-induced hyperalgesia was blocked by apocynin, a NADPH oxidase inhibitor; N(G)-nitro-l-arginine methyl ester, a nonselective NOS inhibitor; and by the potent PN decomposition catalysts (FeTM-4-PyP(5+) and MnTE-2-PyP(5+)). Our findings provide mechanistic insight into the signaling pathways engaged by S1P in the development of hyperalgesia and highlight the contribution of the S1P(1) receptor-to-PN signaling in this process. Sphingosine-1-phosphate (S1P)-induced hyperalgesia is mediated by S1P1 receptor activation and mitigated by inhibition or decomposition of peroxynitrite, providing a target pathway for novel pain management strategies.

Topics: Acetophenones; Anilides; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Hyperalgesia; Lysophospholipids; Male; Metalloporphyrins; NG-Nitroarginine Methyl Ester; Organophosphonates; Oxadiazoles; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Time Factors

Ceramide is a potent pro-inflammatory sphingolipid recently shown to exert potent hyperalgesic responses in rats. Once generated, ceramide is converted by sphingosine kinase (SphK) 1 and/or 2 to one of its active metabolite sphingosine-1-phosphate (S1P), which in turn signals through G-protein coupled S1P receptors. The objectives of this paper were to define whether ceramide-induced hyperalgesia is driven by S1P. Our results show that intraplantar injection of ceramide in rats led to a time-dependent development of thermal hyperalgesia that was associated with an increase in tumor necrosis factor-α (TNF-α) in paw tissues. The development of hyperalgesia was significantly attenuated by a soluble TNF receptor I. TNF-α is known to activate SphK1, thus S1P production, and our results demonstrate that, the development of hyperalgesia was attenuated in a dose-dependent fashion by a well characterized inhibitor of SphK1 and SphK2 (SK-I) and by a murine monoclonal anti-S1P antibody (LT1002). LT1017, the isotype-matched control monoclonal antibody for LT1002, had no effect. Our results further demonstrate that S1P contributes to the development of hyperalgesia via the S1P receptor 1 subtype (S1PR(1)), since responses were blocked by a well characterized S1PR(1) antagonist, W146, but not by its inactive enantiomer, W140. Collectively, these results provide mechanistic evidence implicating the S1P-to-S1PR(1) pathway as a downstream signaling pathway in ceramide-induced hyperalgesia. Targeting S1P may be a novel therapeutic approach in pain management.

Topics: Anilides; Animals; Ceramides; Hyperalgesia; Inflammation Mediators; Injections, Subcutaneous; Lysophospholipids; Male; Mice; Organophosphonates; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) has been demonstrated to enhance endothelial barrier function in vivo and in vitro. However, different S1P receptor subtypes have been indicated to play different or even opposing roles in the regulation of vascular barrier function. This study aims to differentiate the roles of endogenous endothelial S1P subtype receptors in the regulation of permeability in intact microvessels using specific receptor agonist and antagonists. Microvessel permeability was measured with hydraulic conductivity (L(p)) in individually perfused rat mesenteric venules. S1P-mediated changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura-2-loaded venules. Confocal images of fluorescent immunostaining illustrated the spatial expressions of three S1P subtype receptors (S1P(R1-3)) in rat venules. The application of S1P (1 μM) in the presence of S1P(R1-3) inhibited platelet-activating factor- or bradykinin-induced permeability increase. This S1P effect was reversed only with a selective S1P(R1) antagonist, W-146, and was not affected by S1P(R2) or S1P(R3) antagonists JTE-013 and CAY-10444, respectively. S1P(R1) was also identified as the sole receptor responsible for S1P-mediated increases in endothelial [Ca(2+)](i). S1P(R2) or S1P(R3) antagonist alone affected neither basal L(p) nor platelet-activating factor-induced permeability increase. The selective S1P(R1) agonist, SEW-2871, showed similar [Ca(2+)](i) and permeability effect to that of S1P. These results indicate that, despite the presence of S1P(R1-3) in the intact venules, only the activation of endothelial S1P(R1) is responsible for the protective action of S1P on microvessel permeability and that endogenous S1P(R2) or S1P(R3) did not exhibit functional roles in the regulation of permeability under basal or acutely stimulated conditions.

Topics: Anilides; Animals; Calcium; Capillary Permeability; Endothelium, Vascular; Female; Lysophospholipids; Mesentery; Models, Animal; Organophosphonates; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Venules

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

Topics: Anilides; Animals; Capillary Permeability; Cells, Cultured; CHO Cells; Cricetinae; Disease Models, Animal; Evans Blue; Humans; Lymph Nodes; Lymphocytes; Lysophospholipids; Mice; Mice, Inbred C57BL; Models, Biological; Organophosphonates; Phenotype; Pulmonary Edema; Receptors, Lysosphingolipid; Sphingosine; Stereoisomerism

Topics: Anilides; Animals; Binding Sites; Humans; Lysophospholipids; Models, Biological; Organophosphonates; Phenotype; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine