ABSTRACT
Asthma accounts for 380,000 deaths a year. Carotid body denervation has been shown to have a profound effect on airway hyper-responsiveness in animal models but a mechanistic explanation is lacking. Here we demonstrate, using a rat model of asthma (OVA-sensitized), that carotid body activation during airborne allergic provocation is caused by systemic release of lysophosphatidic acid (LPA). Carotid body activation by LPA involves TRPV1 and LPA-specific receptors, and induces parasympathetic (vagal) activity. We demonstrate that this activation is sufficient to cause acute bronchoconstriction. Moreover, we show that prophylactic administration of TRPV1 (AMG9810) and LPA (BrP-LPA) receptor antagonists prevents bradykinin-induced asthmatic bronchoconstriction and, if administered following allergen exposure, reduces the associated respiratory distress. Our discovery provides mechanistic insight into the critical roles of carotid body LPA receptors in allergen-induced respiratory distress and suggests alternate treatment options for asthma.
Subject(s)
Acrylamides/therapeutic use , Asthma/prevention & control , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Carotid Body/metabolism , Lysophospholipids/therapeutic use , Receptors, Lysophosphatidic Acid/metabolism , TRPV Cation Channels/metabolism , Acrylamides/pharmacology , Animals , Asthma/etiology , Asthma/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Disease Models, Animal , Drug Evaluation, Preclinical , Lysophospholipids/pharmacology , Male , Rats, Inbred BN , Rats, Sprague-Dawley , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , TRPV Cation Channels/antagonists & inhibitorsABSTRACT
Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the ß-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the ß-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.
Subject(s)
Catecholamines/metabolism , Motor Activity/drug effects , Plant Extracts/pharmacology , Receptors, Lysophosphatidic Acid/metabolism , Adrenal Glands/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Blood Glucose/metabolism , Epinephrine/blood , Fasting , Glycogenolysis , Male , Mice, Inbred BALB C , Mice, Inbred ICR , Norepinephrine/blood , Physical Conditioning, Animal , Physical Endurance/drug effects , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Rotarod Performance TestABSTRACT
OBJECTIVE: To investigate the effect of lysophosphatidic acid (LPA) receptor inhibition in a mouse model of autoantibody-mediated arthritis. METHODS: Arthritis was induced in C57BL/6 mice by K/BxN serum transfer. Arthritic mice were treated with the LPA receptor antagonist, Ki16425 and arthritis severity was assessed clinically and histologically. Expression of inflammatory mediators in joints was identified by a mouse cytokine array and validated by western blot and real-time PCR assays. Effects of treatment with LPA receptor antagonist or with small interfering RNA on bone metabolism were assessed by in vitro assays of osteoclastogenesis, bone resorption, osteoblasts differentiation and bone mineralisation. RESULTS: Mice treated with the LPA receptor antagonist Ki16425 showed attenuated arthritis characterised by reduction of synovial inflammation, cartilage damage and, more markedly, bone erosion. We detected increased apoptosis, reduction of inflammatory mediators and of bone remodelling proteins in arthritic joints from mice treated with Ki16425. In addition, we demonstrated that inhibition or suppression of LPA1 receptor reduces osteoclast differentiation and bone resorption and, on the contrary, it promotes differentiation of osteoblasts and bone mineralisation. CONCLUSIONS: Pharmacological inhibition of LPA1 receptor in the K/BxN serum-transfer arthritis model led to reduction of severity of arthritis involving multiple mechanisms, increased apoptosis, reduced inflammatory mediators and proteins involved in bone remodelling, that show LPA1 as a very promising target in rheumatoid arthritis treatment.
Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , Isoxazoles/pharmacology , Propionates/pharmacology , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Synovitis/drug therapy , Animals , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Autoantibodies/immunology , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Osteoblasts/drug effects , Osteoclasts/drug effects , Synovitis/immunologyABSTRACT
Lysophosphatidic acid (LPA) is a class of bioactive phospholipid that displays a wide range of cellular effects via LPA receptors, of which six have been identified (LPAR1-6). In serum and plasma, LPA production occurs mainly by the hydrolysis of lysophosphatidylcholine by the phospholipase D activity of autotaxin (ATX). The involvement of the LPA pathway in driving chronic wound-healing conditions, such as idiopathic pulmonary fibrosis, has suggested targets in this pathway could provide potential therapeutic approaches. Mice with LPAR1 knockout or tissue-specific ATX deletion have demonstrated reduced lung fibrosis following bleomycin challenge. Therefore, strategies aimed at antagonizing LPA receptors or inhibiting ATX have gained considerable attention. This Review will summarize the current status of identifying small-molecule modulators of the LPA pathway. The therapeutic utility of LPA modulators for the treatment of fibrotic diseases will soon be revealed as clinical trials are already in progress in this area.
Subject(s)
Lysophospholipids/metabolism , Pulmonary Fibrosis/drug therapy , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Small Molecule Libraries/therapeutic use , Animals , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Phosphorous Acids/chemistry , Phosphorous Acids/therapeutic use , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Small Molecule Libraries/chemistryABSTRACT
Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s)were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 >LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitorY-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.
Subject(s)
Endothelium, Vascular/drug effects , GTP-Binding Proteins/metabolism , Glycoproteins/pharmacology , Plant Extracts/chemistry , Animals , Calcium Signaling/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Expression Profiling , Glycoproteins/chemistry , Humans , Isoxazoles/pharmacology , Lysophospholipids/chemistry , Molecular Targeted Therapy , Mutagenesis, Site-Directed , Mutation/genetics , PC12 Cells , Panax , Pertussis Toxin/pharmacology , Plant Proteins/chemistry , Propionates/pharmacology , Protein Binding/genetics , Rats , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Transgenes/geneticsABSTRACT
CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Drug Delivery Systems/methods , Macromolecular Substances/antagonists & inhibitors , Animals , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Drug Discovery/methods , Drug Evaluation, Preclinical , High-Throughput Screening Assays/methods , Humans , Indoles/pharmacology , Macromolecular Substances/metabolism , Models, Biological , Phenylpropionates/pharmacology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Phosphoproteins/physiology , Protein Binding/drug effects , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/metabolism , Receptors, Lysophosphatidic Acid/physiology , Small Molecule Libraries/pharmacology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/metabolism , Sodium-Hydrogen Exchangers/physiology , SwineABSTRACT
Radiation pneumonitis (RP) is a serious complication of radiation therapy for thoracic tumors. Lysophosphatidic acid (LPA) and its receptors LPAâ were reported to participate in the processes of inflammation. We tested the hypothesis that LPA and its receptors LPAâ , take part in the pathogenesis of RP. In our study, irradiation increased LPA levels in the lung and expression of LPAâ . To further determine the role of LPAâ , we performed pharmacological knockout of LPAâ by a specific antagonist, VPC-12249. On day 60 post-irradiation, RP was significantly alleviated in a dose-dependent manner in mice treated with VPC-12249, as shown by H&E staining, malondialdehyde (MDA, an indicator of oxidative damage) assay in lung, and concentrations of proinflammatory and profibrotic cytokines in plasma, including IL-1ß, TNF-α, and TGF-ß1. Additionally, VPC-12249 administration decreased the phosphorylation of IκB-α (the initial event that activates the NF-κB signal way), and expression of TGF-ß1, CTGF, and α-SMA mRNA. Our findings suggest that LPA and LPAâ may play a pivotal role in RP, and LPA-LPAâ may serve as novel therapeutic targets for the treatment of RP.
Subject(s)
Lysophospholipids/pharmacology , Radiation Pneumonitis/drug therapy , Radiation Pneumonitis/etiology , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/physiology , Animals , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Lung/drug effects , Lung/metabolism , Lung/pathology , Lysophospholipids/metabolism , Mice , Mice, Inbred C57BL , Oleic Acids/administration & dosage , Oleic Acids/pharmacology , Oleic Acids/therapeutic use , Organophosphates/administration & dosage , Organophosphates/pharmacology , Organophosphates/therapeutic use , Phosphorylation/drug effects , Radiation Pneumonitis/metabolism , Radiation-Protective Agents/administration & dosage , Radiation-Protective Agents/pharmacology , Radiation-Protective Agents/therapeutic use , Receptors, Lysophosphatidic Acid/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
Lysophosphatidic acid (LPA) is a simple lipid with many important biological functions such as the regulation of cellular proliferation, cellular migration, differentiation, and suppression of apoptosis. Although a direct angiogenic effect of LPA has not been reported to date, there are indications that LPA promotes angiogenesis. In addition, LPA is a chemoattractant for cultured endothelial cells and promotes barrier function in such cultures. To test the hypothesis that LPA is angiogenic, we used the chicken chorio-allantoic membrane (CAM) assay. Sequence analysis of the cloned, full-length chicken LPA receptor cDNAs revealed three receptor types that are orthologous to the mammalian LPA(1), LPA(2), and LPA(3) receptors. We document herein that LPA is angiogenic in the CAM system and further that synthetic LPA receptor agonists and antagonists mimic or block this response, respectively. Our results predict that LPA receptor antagonists are a possible therapeutic route to interdicting angiogenesis.
Subject(s)
Lysophospholipids/pharmacology , Neovascularization, Physiologic/drug effects , Receptors, Lysophosphatidic Acid/physiology , Amino Acid Sequence , Angiogenesis Inhibitors/pharmacology , Animals , Chick Embryo , Drug Evaluation, Preclinical , Molecular Sequence Data , Organophosphates/pharmacology , Pyridines/pharmacology , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Sequence Homology, Amino Acid , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Substrate Specificity , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Darmstoff describes a family of gut smooth muscle-stimulating acetal phosphatidic acids initially isolated and characterized from the bath fluid of stimulated gut over 50 years ago. Despite similar structural and biological profiles, Darmstoff analogs have not previously been examined as potential LPA mimetics. Here, we report a facile method for the synthesis of potassium salts of Darmstoff analogs. To understand the effect of stereochemistry on lysophosphatidic acid mimetic activity, synthesis of optically pure stereoisomers of selected Darmstoff analogs was achieved starting with chiral methyl glycerates. Each Darmstoff analog was evaluated for subtype-specific LPA receptor agonist/antagonist activity, PPARgamma activation, and autotaxin inhibition. From this study we identified compound 12 as a pan-antagonist and several pan-agonists for the LPA(1-3) receptors. Introduction of an aromatic ring in the lipid chain such as analog 22 produced a subtype-specific LPA(3) agonist with an EC(50) of 692 nM. Interestingly, regardless of their LPA(1/2/3) ligand properties all of the Darmstoff analogs tested activated PPARgamma. However, these compounds are weak inhibitors of autotaxin. The results indicate that Darmstoff analogs constitute a novel class of lysophosphatidic acid mimetics.