Science.gov

Sample records for acid receptor gpr40

  1. [Antinociceptive effect of docosahexaenoic acid (DHA) through long fatty acid receptor G protein-coupled receptor 40 (GPR40)].

    PubMed

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Tokuyama, Shogo

    2014-01-01

    Fatty acids, one class of essential nutrients for humans, are an important source of energy and an essential component of cell membranes. They also function as signal transduction molecules in a variety of biological phenomena. The important functional role of fatty acids in both onset and suppression of pain has become increasingly apparent in recent years. Recently, we have also demonstrated that the release of an endogenous opioid peptide, β-endorphin, plays an important role in the induction of docosahexaenoic acid (DHA)-induced antinociception. It is well known that fatty acids affect intracellular and intercellular signaling as well as the membrane fluidity of neurons. In addition to intracellular actions, unbound free fatty acids (FFAs) can also carry out extracellular signaling by stimulating the G protein-coupled receptor (GPCR). Among these receptors, G protein-coupled receptor 40 (GPR40) has been reported to be activated by long-chain fatty acids such as DHA, eicosapentaenoic acid (EPA) and arachidonic acid. In the peripheral area, GPR40 is preferentially expressed in pancreatic β-cells and is known to relate to the secretion of hormone and peptides. On the other hand, even though this receptor is widely distributed in the central nervous system, reports studying the role and functions of GPR40 in the brain have not been found. In this review, we summarize the findings of our recent study about the long-chain fatty acid receptor GPR40 as a novel pain regulatory system. PMID:24584021

  2. The Free Fatty Acid Receptor G Protein-coupled Receptor 40 (GPR40) Protects from Bone Loss through Inhibition of Osteoclast Differentiation*

    PubMed Central

    Wauquier, Fabien; Philippe, Claire; Léotoing, Laurent; Mercier, Sylvie; Davicco, Marie-Jeanne; Lebecque, Patrice; Guicheux, Jérôme; Pilet, Paul; Miot-Noirault, Elisabeth; Poitout, Vincent; Alquier, Thierry; Coxam, Véronique; Wittrant, Yohann

    2013-01-01

    The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40−/− mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/β) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40−/− primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40−/− mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications. PMID:23335512

  3. Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca2+ signaling through activation of free fatty acid receptor GPR40.

    PubMed

    Kruska, Nicol; Reiser, Georg

    2011-08-01

    The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

  4. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875.

    PubMed

    Srivastava, Ankita; Yano, Jason; Hirozane, Yoshihiko; Kefala, Georgia; Gruswitz, Franz; Snell, Gyorgy; Lane, Weston; Ivetac, Anthony; Aertgeerts, Kathleen; Nguyen, Jasmine; Jennings, Andy; Okada, Kengo

    2014-09-01

    Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.

  5. Differential Gene Expression in GPR40-Overexpressing Pancreatic β-cells Treated with Linoleic Acid

    PubMed Central

    Kim, In-Su; Yang, So-Young; Han, Joo-Hui; Jung, Sang-Hyuk; Park, Hyun-Soo

    2015-01-01

    "G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic β-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 µM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus. PMID:25729276

  6. Free fatty acid receptor 1 (FFAR1/GPR40) signaling affects insulin secretion by enhancing mitochondrial respiration during palmitate exposure.

    PubMed

    Kristinsson, Hjalti; Bergsten, Peter; Sargsyan, Ernest

    2015-12-01

    Fatty acids affect insulin secretion via metabolism and FFAR1-mediated signaling. Recent reports indicate that these two pathways act synergistically. Still it remains unclear how they interrelate. Taking into account the key role of mitochondria in insulin secretion, we attempted to dissect the metabolic and FFAR1-mediated effects of fatty acids on mitochondrial function. One-hour culture of MIN6 cells with palmitate significantly enhanced mitochondrial respiration. Antagonism or silencing of FFAR1 prevented the palmitate-induced rise in respiration. On the other hand, in the absence of extracellular palmitate FFAR1 agonists caused a modest increase in respiration. Using an agonist of the M3 muscarinic acetylcholine receptor and PKC inhibitor we found that in the presence of the fatty acid mitochondrial respiration is regulated via Gαq protein-coupled receptor signaling. The increase in respiration in palmitate-treated cells was largely due to increased glucose utilization and oxidation. However, glucose utilization was not dependent on FFAR1 signaling. Collectively, these results indicate that mitochondrial respiration in palmitate-treated cells is enhanced via combined action of intracellular metabolism of the fatty acid and the Gαq-coupled FFAR1 signaling. Long-term palmitate exposure reduced ATP-coupling efficiency of mitochondria and deteriorated insulin secretion. The presence of the FFAR1 antagonist during culture did not improve ATP-coupling efficiency, however, it resulted in enhanced mitochondrial respiration and improved insulin secretion after culture. Taken together, our study demonstrates that during palmitate exposure, integrated actions of fatty acid metabolism and fatty acid-induced FFAR1 signaling on mitochondrial respiration underlie the synergistic action of the two pathways on insulin secretion.

  7. CoMSIA study on substituted aryl alkanoic acid analogs as GPR40 agonists.

    PubMed

    Bhatt, Aaditya; Patel, Pallav D; Patel, Maulik R; Singh, Satyakam; Lau-Cam, Cesar A; Talele, Tanaji T

    2011-05-01

    GPR40, a G-protein-coupled receptor has been well established to play a crucial role in regulating blood glucose levels. Hence, GPR40 is a potential target for future antidiabetic agents. The present 3D QSAR study is aimed at delineating structural parameters governing GPR40 agonistic activity. To meet this objective, a comparative molecular similarity indices analysis for 63 different GPR40 agonists was performed using two methods; a ligand-based 3D QSAR model employing the atom fit alignment method and a receptor-based 3D QSAR model that was derived from the predicted binding conformations obtained by docking all the GPR40 agonists at the active site of GPR40. The results of these studies showed the ligand-based model to be superior (r(cv)(2) value of 0.610) to the receptor-based model (r(cv)(2) value of 0.519) in terms of statistical data. The predictive ability of these models was evaluated using a test set of 15 compounds not included in the preliminary training set of 48 compounds. The predictive r(2) values for the ligand- and the receptor-based models were found to be 0.863 and 0.599, respectively. Further, interpretation of the comparative molecular similarity indices analysis contour maps with reference to the active site of GPR40 provided an insight into GPR40-agonist interactions. PMID:21352503

  8. CoMSIA study on substituted aryl alkanoic acid analogs as GPR40 agonists.

    PubMed

    Bhatt, Aaditya; Patel, Pallav D; Patel, Maulik R; Singh, Satyakam; Lau-Cam, Cesar A; Talele, Tanaji T

    2011-05-01

    GPR40, a G-protein-coupled receptor has been well established to play a crucial role in regulating blood glucose levels. Hence, GPR40 is a potential target for future antidiabetic agents. The present 3D QSAR study is aimed at delineating structural parameters governing GPR40 agonistic activity. To meet this objective, a comparative molecular similarity indices analysis for 63 different GPR40 agonists was performed using two methods; a ligand-based 3D QSAR model employing the atom fit alignment method and a receptor-based 3D QSAR model that was derived from the predicted binding conformations obtained by docking all the GPR40 agonists at the active site of GPR40. The results of these studies showed the ligand-based model to be superior (r(cv)(2) value of 0.610) to the receptor-based model (r(cv)(2) value of 0.519) in terms of statistical data. The predictive ability of these models was evaluated using a test set of 15 compounds not included in the preliminary training set of 48 compounds. The predictive r(2) values for the ligand- and the receptor-based models were found to be 0.863 and 0.599, respectively. Further, interpretation of the comparative molecular similarity indices analysis contour maps with reference to the active site of GPR40 provided an insight into GPR40-agonist interactions.

  9. A Gut Microbial Metabolite of Linoleic Acid, 10-Hydroxy-cis-12-octadecenoic Acid, Ameliorates Intestinal Epithelial Barrier Impairment Partially via GPR40-MEK-ERK Pathway*

    PubMed Central

    Miyamoto, Junki; Mizukure, Taichi; Park, Si-Bum; Kishino, Shigenobu; Kimura, Ikuo; Hirano, Kanako; Bergamo, Paolo; Rossi, Mauro; Suzuki, Takuya; Arita, Makoto; Ogawa, Jun; Tanabe, Soichi

    2015-01-01

    Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca2+]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease. PMID:25505251

  10. GPR40 receptor activation leads to CREB phosphorylation and improves cognitive performance in an Alzheimer's disease mouse model.

    PubMed

    Khan, Muhammad Zahid; Zhuang, Xuxu; He, Ling

    2016-05-01

    Alzheimer's disease (AD) is a very complex neurodegenerative disorder as neuronal loss is a prominent and initial feature of AD. This loss correlates with cognitive deficits more closely than amyloid load. GPR40 receptor belongs to the class of G-protein coupled receptors, is expressed in wide parts of the brain including the hippocampus which is involved in spatial learning and memory. Till now, there are few studies investigating the functional role of GPR40 in brain. In this study, we evaluated the functional role of GPR40 receptor in the A-beta AD mice model. Administration of Aβ1-42 (410pmol) intracerebroventricularly (i.c.v.) once at the beginning of experiment significantly impaired cognitive performance (in step-through passive test), the ability of spatial learning and memory in (Morris water maze test), working memory, attention, anxiety in (Novel object recognition test), and spatial working and reference-memory in (Hole board discrimination test) compared with the control group. The results revealed that GPR40 receptor treatment groups significantly ameliorated model mice cognitive performance. All GPR40 receptor agonist GW9508, treatment groups enhanced the learning and memory ability in Step-through passive test, Morris water maze test, Hole board discrimination test, Novel object recognition test. Furthermore, we have observed that activation of GPR40 receptor provoked the phosphorylation of the cAMP response element binding protein (CREB) and significant increase in neurotropic factors including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrohin-4 (NT-4) in mouse hippocampal neurons and contribute to neurogenesis. These results suggest that GPR40 is a suitable therapeutic candidate for neurogenesis and neuroprotection in the treatment and prevention of AD.

  11. Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

    PubMed

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty

  12. Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

    PubMed Central

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty

  13. Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism.

    PubMed

    Puebla, Carlos; Cisterna, Bruno A; Salas, Daniela P; Delgado-López, Fernando; Lampe, Paul D; Sáez, Juan C

    2016-05-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.

  14. Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets

    SciTech Connect

    Alquier, Thierry; Peyot, Marie-Line; Latour, M. G.; Kebede, Melkam; Sorensen, Christina M.; Gesta, Stephane; Kahn, C. R.; Smith, Richard D.; Jetton, Thomas L.; Metz, Thomas O.; Prentki, Marc; Poitout, Vincent J.

    2009-11-01

    The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

  15. Potentiation of Glucose-stimulated Insulin Secretion by the GPR40–PLC–TRPC Pathway in Pancreatic β-Cells

    PubMed Central

    Yamada, Hodaka; Yoshida, Masashi; Ito, Kiyonori; Dezaki, Katsuya; Yada, Toshihiko; Ishikawa, San-e; Kakei, Masafumi

    2016-01-01

    G protein-coupled receptors (GPCRs) are expressed in pancreatic beta-cells. G protein-coupled receptor 40 (GPR40) contributes to medium- or long-chain fatty acid-induced amplification of glucose-stimulated insulin secretion (GSIS), and GPR40 agonists are promising therapeutic targets in type 2 diabetes. Recently, we demonstrated that glucagon-like peptide 1, a ligand of pancreatic GPCR, activates a class of nonselective cation channels (NSCCs) and enhances GSIS. The aim of the current study was to determine whether the GPR40 signal interacts with NSCCs. A GPR40 agonist (fasiglifam) potentiated GSIS at 8.3 and 16.7 mM glucose but not 2.8 mM glucose. The NSCC current was activated by fasiglifam at 5.6 mM glucose with 100 μM tolbutamide (−70 mV), and this activation was prevented by the presence of pyrazole-3 (transient receptor potential canonical; a TRPC3 channel blocker). Inhibitors of phospholipase C or protein kinase C (PKC) inhibited the increases in GSIS and the NSCC current induced by GPR40 stimulation. The present study demonstrates a novel mechanism for the regulation of insulin secretion by GPR40 agonist in pancreatic beta-cells. The stimulation of the GPR40–PLC/PKC–TRPC3 channel pathway potentiates GSIS by the depolarization of the plasma membrane in pancreatic beta-cell. PMID:27180622

  16. Activation of tongue-expressed GPR40 and GPR120 by non caloric agonists is not sufficient to drive preference in mice.

    PubMed

    Godinot, N; Yasumatsu, K; Barcos, M E; Pineau, N; Ledda, M; Viton, F; Ninomiya, Y; le Coutre, J; Damak, S

    2013-10-10

    There is mounting evidence that, in addition to texture and olfaction, taste plays a role in the detection of long chain fatty acids. Triglycerides, the main components of oils and dietary fat, are hydrolyzed in the mouth by a lingual lipase secreted from the von Ebner gland and the released free fatty acids are detected by the taste system. GPR40 and GPR120, two fatty acid responsive G-protein-coupled receptors (GPCRs), are expressed in taste bud cells, and knockout mice lacking either of those receptors have blunted taste nerve responses to and reduced preference for fatty acids. Here we investigated whether activation of those GPCRs is sufficient to elicit fat taste and preference. Five non-fatty acid agonists of GPR40 and two non-fatty acid agonists of GPR120 activated the glossopharyngeal nerve of wild-type mice but not of knockout mice lacking the cognate receptor. In human subjects, two-alternative forced choice (2-AFC) tests, triangle tests and sensory profiling showed that non fatty acid agonists of GPR40 dissolved in water are detected in sip and spit tests and elicit a taste similar to that of linoleic acid, whereas 2-AFC tests showed that two agonists of GPR120 in water are not perceived fattier than water alone. Wild-type mice did not show any preference for five agonists of GPR40, two agonists of GPR120 and mixtures of both agonists over water in two-bottle preference tests. Together these data indicate that GPR40 mediated taste perception is not sufficient to generate preference. PMID:23831422

  17. Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.

    PubMed

    Ren, Xiao-Min; Cao, Lin-Ying; Zhang, Jing; Qin, Wei-Ping; Yang, Yu; Wan, Bin; Guo, Liang-Hong

    2016-04-01

    Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

  18. Intragastric fat self-administration is impaired in GPR40/120 double knockout mice

    PubMed Central

    Sclafani, Anthony; Touzani, Khalid; Ackroff, Karen

    2015-01-01

    Mice acquire strong preferences for flavors paired with intragastric (IG) fat infusions. This IG fat conditioning is attenuated in double knockout (DoKO) mice missing GPR40 and GPR120 fatty acid receptors. Here we determined if GPR40/120 DoKO mice are also impaired in IG fat self-administration in an operant lick task. In daily 1-h sessions the mice were trained with a sipper spout that contained dry food pellets; licks on the spout triggered infusions of IG fat (Intralipid). The training sessions were followed by test sessions with an empty spout. GPR40/120 DoKO mice self-infused more 20% fat than wild type (WT) C57BL/6 mice in training with a food-baited spout (2.4 vs. 2.0 kcal/h) but self-infused less 20% fat than WT mice in empty spout tests (1.2 vs. 1.7 kcal/h). The DoKO mice also self-infused less 5% fat than WT mice (0.6 vs. 1.3 kcal/h) although both groups emitted more licks for 5% fat than 20% fat. The DoKO and WT mice did not differ, however, in their self-infusion of 12.5% glucose (1.5 vs. 1.6 kcal/h), which is isocaloric to 5% fat. A second 5% IL test showed that the DoKO mice reverted to a reduced self-infusion compared to WT mice. When the infusion was shifted to water, WT mice reduced licking in the first extinction session, whereas DoKO mice were less sensitive to the absence of infused fat. Our results indicate that post-oral GPR40/120 signaling is not required to process IG fat infusions in food-baited spout training sessions but contributes to post-oral fat reinforcement in empty spout tests and flavor conditioning tests. PMID:25911263

  19. Featured Article: Deficiency of G-protein coupled receptor 40, a lipid-activated receptor, heightens in vitro- and in vivo-induced murine osteoarthritis

    PubMed Central

    Philippe, Claire; Mercier, Sylvie; Coxam, Véronique; Wittrant, Yohann

    2015-01-01

    Osteoarthritis (OA) is an age-related degenerative joint disease. To date, its management is focused on symptoms (pain and inflammation). Studies suggest that fatty acids can reduce the expression of inflammatory and catalytic mediators, and improve in vivo joint function. Free fatty acid receptors (FFARs) such as G-protein coupled receptor 40 (GPR40) are proposed as attractive therapeutic targets to counteract inflammation and cartilage degradation observed in OA. This study aims to elucidate the involvement of GPR40 in OA. In this study, we used an in vitro model of OA, and surgically induced OA by ligament transection and partial meniscectomy in wild-type and GPR40 deficient mice. OA phenotype was investigated in vivo by histology and genes expression. We demonstrate that IL-1β-treated GPR40−/− chondrocytes secret more inflammatory mediators (nitric oxide, interleukin-6, prostaglandin E2) and active catabolic enzymes (metalloproteinase-2, -9 [MMP-2, MMP-9]), and show decreased anabolism (glycoaminoglycan) compared to GPR40+/+ cells. In accordance with these results, we show that GPR40−/− mice exhibit an aggravated OA-induced phenotype characterized by higher tidemark exposure, frequency of osteophyte formation and subchondral bone sclerosis. Altogether our results demonstrate that GPR40 deficiency leads to an extended OA phenotype, providing evidence that increasing GPR40 activity, by natural or synthetic ligands, could be a new strategy in the management of OA. PMID:25585625

  20. Nutritional Signaling via Free Fatty Acid Receptors.

    PubMed

    Miyamoto, Junki; Hasegawa, Sae; Kasubuchi, Mayu; Ichimura, Atsuhiko; Nakajima, Akira; Kimura, Ikuo

    2016-01-01

    Excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises. Dysfunction of energy balance by excess food intake leads to metabolic diseases, such as obesity and diabetes. Free fatty acids (FFAs) provided by dietary fat are not only important nutrients, but also contribute key physiological functions via FFA receptor (FFAR)-mediated signaling molecules, which depend on FFAs' carbon chain length and the ligand specificity of the receptors. Functional analyses have revealed that FFARs are critical for metabolic functions, such as peptide hormone secretion and inflammation, and contribute to energy homeostasis. In particular, recent studies have shown that the administration of selective agonists of G protein-coupled receptor (GPR) 40 and GPR120 improved glucose metabolism and systemic metabolic disorders. Furthermore, the anti-inflammation and energy metabolism effects of short chain FAs have been linked to the activation of GPR41 and GPR43. In this review, we summarize recent progress in research on FFAs and their physiological roles in the regulation of energy metabolism. PMID:27023530

  1. Nutritional Signaling via Free Fatty Acid Receptors

    PubMed Central

    Miyamoto, Junki; Hasegawa, Sae; Kasubuchi, Mayu; Ichimura, Atsuhiko; Nakajima, Akira; Kimura, Ikuo

    2016-01-01

    Excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises. Dysfunction of energy balance by excess food intake leads to metabolic diseases, such as obesity and diabetes. Free fatty acids (FFAs) provided by dietary fat are not only important nutrients, but also contribute key physiological functions via FFA receptor (FFAR)-mediated signaling molecules, which depend on FFAs’ carbon chain length and the ligand specificity of the receptors. Functional analyses have revealed that FFARs are critical for metabolic functions, such as peptide hormone secretion and inflammation, and contribute to energy homeostasis. In particular, recent studies have shown that the administration of selective agonists of G protein-coupled receptor (GPR) 40 and GPR120 improved glucose metabolism and systemic metabolic disorders. Furthermore, the anti-inflammation and energy metabolism effects of short chain FAs have been linked to the activation of GPR41 and GPR43. In this review, we summarize recent progress in research on FFAs and their physiological roles in the regulation of energy metabolism. PMID:27023530

  2. Potentiation of insulin secretion and improvement of glucose intolerance by combining a novel G protein-coupled receptor 40 agonist DS-1558 with glucagon-like peptide-1 receptor agonists.

    PubMed

    Nakashima, Ryutaro; Yano, Tatsuya; Ogawa, Junko; Tanaka, Naomi; Toda, Narihiro; Yoshida, Masao; Takano, Rieko; Inoue, Masahiro; Honda, Takeshi; Kume, Shoen; Matsumoto, Koji

    2014-08-15

    G protein-coupled receptor 40 (GPR40) is a Gq-coupled receptor for free fatty acids predominantly expressed in pancreatic β-cells. In recent years, GPR40 agonists have been investigated for use as novel therapeutic agents in the treatment of type 2 diabetes. We discovered a novel small molecule GPR40 agonist, (3S)-3-ethoxy-3-(4-{[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]oxy}phenyl)propanoic acid (DS-1558). The GPR40-mediated effects of DS-1558 on glucose-stimulated insulin secretion were evaluated in isolated islets from GPR40 knock-out and wild-type (littermate) mice. The GPR40-mediated effects on glucose tolerance and insulin secretion were also confirmed by an oral glucose tolerance test in these mice. Furthermore, oral administration of DS-1558 (0.03, 0.1 and 0.3mg/kg) significantly and dose-dependently improved hyperglycemia and increased insulin secretion during the oral glucose tolerance test in Zucker fatty rats, the model of insulin resistance and glucose intolerance. Next, we examined the combination effects of DS-1558 with glucagon like peptide-1 (GLP-1). DS-1558 not only increased the glucose-stimulated insulin secretion by GLP-1 but also potentiated the maximum insulinogenic effects of GLP-1 after an intravenous glucose injection in normal Sprague Dawley rats. Furthermore, the glucose lowering effects of exendin-4, a GLP-1 receptor agonist, were markedly potentiated by the DS-1558 (3mg/kg) add-on in diabetic db/db mice during an intraperitoneal glucose tolerance test. In conclusion, our results indicate that add-on GPR40 agonists to GLP-1 related agents might be a potential treatment compared to single administration of these compounds. Therefore the combinations of these agents are a novel therapeutic option for type 2 diabetes.

  3. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

    PubMed

    Mancini, Arturo D; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-08-21

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

  4. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1*

    PubMed Central

    Mancini, Arturo D.; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-01-01

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. PMID:26157145

  5. Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine

    PubMed Central

    Christensen, Louise W; Kuhre, Rune E; Janus, Charlotte; Svendsen, Berit; Holst, Jens J

    2015-01-01

    Glucagon-like peptide 1 (GLP-1) plays a central role in modern treatment of type 2 diabetes (T2DM) in the form of GLP-1 enhancers and GLP-1 mimetics. An alternative treatment strategy is to stimulate endogenous GLP-1 secretion from enteroendocrine L cells using a targeted approach. The G-protein-coupled receptor, FFAR1 (previously GPR40), expressed on L cells and activated by long-chain fatty acids (LCFAs) is a potential target. A link between FFAR1 activation and GLP-1 secretion has been demonstrated in cellular models and small-molecule FFAR1 agonists have been developed. In this study, we examined the effect of FFAR1 activation on GLP-1 secretion using isolated, perfused small intestines from rats, a physiologically relevant model allowing distinction between direct and indirect effects of FFAR1 activation. The endogenous FFAR1 ligand, linoleic acid (LA), and four synthetic FFAR1 agonists (TAK-875, AMG 837, AM-1638, and AM-5262) were administered through intraluminal and intra-arterial routes, respectively, and dynamic changes in GLP-1 secretion were evaluated. Vascular administration of 10 μmol/L TAK-875, 10 μmol/L AMG 837, 1 μmol/L and 0.1 μmol/L AM-1638, 1 μmol/L AM-6252, and 1 mmol/L LA, all significantly increased GLP-1 secretion compared to basal levels (P < 0.05), whereas luminal administration of LA and FFAR1 agonists was ineffective. Thus, both natural and small-molecule agonists of the FFAR1 receptor appear to require absorption prior to stimulating GLP-1 secretion, indicating that therapies based on activation of nutrient sensing may be more complex than hitherto expected. PMID:26381015

  6. Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine.

    PubMed

    Christensen, Louise W; Kuhre, Rune E; Janus, Charlotte; Svendsen, Berit; Holst, Jens J

    2015-09-01

    Glucagon-like peptide 1 (GLP-1) plays a central role in modern treatment of type 2 diabetes (T2DM) in the form of GLP-1 enhancers and GLP-1 mimetics. An alternative treatment strategy is to stimulate endogenous GLP-1 secretion from enteroendocrine L cells using a targeted approach. The G-protein-coupled receptor, FFAR1 (previously GPR40), expressed on L cells and activated by long-chain fatty acids (LCFAs) is a potential target. A link between FFAR1 activation and GLP-1 secretion has been demonstrated in cellular models and small-molecule FFAR1 agonists have been developed. In this study, we examined the effect of FFAR1 activation on GLP-1 secretion using isolated, perfused small intestines from rats, a physiologically relevant model allowing distinction between direct and indirect effects of FFAR1 activation. The endogenous FFAR1 ligand, linoleic acid (LA), and four synthetic FFAR1 agonists (TAK-875, AMG 837, AM-1638, and AM-5262) were administered through intraluminal and intra-arterial routes, respectively, and dynamic changes in GLP-1 secretion were evaluated. Vascular administration of 10 μmol/L TAK-875, 10 μmol/L AMG 837, 1 μmol/L and 0.1 μmol/L AM-1638, 1 μmol/L AM-6252, and 1 mmol/L LA, all significantly increased GLP-1 secretion compared to basal levels (P < 0.05), whereas luminal administration of LA and FFAR1 agonists was ineffective. Thus, both natural and small-molecule agonists of the FFAR1 receptor appear to require absorption prior to stimulating GLP-1 secretion, indicating that therapies based on activation of nutrient sensing may be more complex than hitherto expected.

  7. Treatment with CNX-011-67, a novel GPR40 agonist, delays onset and progression of diabetes and improves beta cell preservation and function in male ZDF rats

    PubMed Central

    2013-01-01

    Background The role of G protein-coupled receptor (GPR40), which is highly expressed in pancreatic beta cells, has been studied extensively in the amelioration of beta cell dysfunction in T2D using rat and mouse islets, beta cell lines and in animal models of diabetes. But its potential as a therapeutic target has not been fully explored. This aim of the study is to evaluate the therapeutic potential of CNX-011-67, a highly selective, potent and orally bioavailable GPR40 agonist, in controlling diabetes and other metabolic parameters. Methods Seven week old male ZDF rats were treated with either vehicle or CNX-011-67, 5 mg/kg twice daily, for seven weeks. The animals were subjected to oral glucose tolerance and insulin tolerance tests. Plasma glucose, insulin, triglyceride, HbA1c, fructosamine and free fatty acids were measured at selected time points. Pancreas from control and treated animals were subjected to insulin and pancreatic and duodenal homeobox 1 (PDX1) immunohistochemistry and were also evaluated by electron microscopy. Also the potential impact of CNX-011-67 on islet insulin secretion, content, ATP levels and markers of both glucose oxidation, beta cell health in rat islets under chronic glucolipotoxic conditions was evaluated. Results Treatment of male ZDF rats with CNX-011-67 for 7 weeks significantly enhanced insulin secretion in response to oral glucose load, delayed the onset of fasting hyperglycemia by 3 weeks, reduced nonfasting glucose excursions, fasting free fatty acids and triglyceride levels. A significant increase in PDX1 expression and insulin content and reduction in plasma fructosamine, HOMA-IR, and beta cell apoptosis were observed. CNX-011-67 improves glucose mediated insulin secretion, insulin gene transcription and islet insulin content in cultured rat islets under chronic glucolipotoxic condition. Also enhanced glucose oxidation in the form of increased islet ATP content and overall improvement in beta cell health in the form of

  8. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

    PubMed Central

    Hopkins, Mandi M.; Zhang, Zhihong; Liu, Ze; Meier, Kathryn E.

    2016-01-01

    Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor. PMID:26821052

  9. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  10. Fasiglifam (TAK-875) has dual potentiating mechanisms via Gαq-GPR40/FFAR1 signaling branches on glucose-dependent insulin secretion.

    PubMed

    Sakuma, Kensuke; Yabuki, Chiori; Maruyama, Minoru; Abiru, Akiko; Komatsu, Hidetoshi; Negoro, Nobuyuki; Tsujihata, Yoshiyuki; Takeuchi, Koji; Habata, Yugo; Mori, Masaaki

    2016-06-01

    Fasiglifam (TAK-875) is a free fatty acid receptor 1 (FFAR1)/G-protein-coupled receptor 40 (GPR40) agonist that improves glycemic control in type 2 diabetes with minimum risk of hypoglycemia. Fasiglifam potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells glucose dependently, although the precise mechanism underlying the glucose dependency still remains unknown. Here, we investigated key cross-talk between the GSIS pathway and FFAR1 signaling, and Ca(2+) dynamics using mouse insulinoma MIN6 cells. We demonstrated that the glucose-dependent insulinotropic effect of fasiglifam required membrane depolarization and that fasiglifam induced a glucose-dependent increase in intracellular Ca(2+) level and amplification of Ca(2+) oscillations. This differed from the sulfonylurea glimepiride that induced changes in Ca(2+) dynamics glucose independently. Stimulation with cell-permeable analogs of IP3 or diacylglycerol (DAG), downstream second messengers of Gαq-FFAR1, augmented GSIS similar to fasiglifam, indicating their individual roles in the potentiation of GSIS pathway. Intriguingly, the IP3 analog triggered similar Ca(2+) dynamics to fasiglifam, whereas the DAG analog had no effect. Despite the lack of an effect on Ca(2+) dynamics, the DAG analog elicited synergistic effects on insulin secretion with Ca(2+) influx evoked by an L-type voltage-dependent calcium channel opener that mimics glucose-dependent Ca(2+) dynamics. These results indicate that the Gαq signaling activated by fasiglifam enhances GSIS pathway via dual potentiating mechanisms in which IP3 amplifies glucose-induced Ca(2+) oscillations and DAG/protein kinase C (PKC) augments downstream secretory mechanisms independent of Ca(2+) oscillations. PMID:27433346

  11. Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.

    PubMed

    Christiansen, Elisabeth; Hudson, Brian D; Hansen, Anders Højgaard; Milligan, Graeme; Ulven, Trond

    2016-05-26

    The free fatty acid receptor 1 (FFA1/GPR40) is a potential target for treatment of type 2 diabetes. Although several potent agonists have been described, there remains a strong need for suitable tracers to interrogate ligand binding to this receptor. We address this by exploring fluorophore-tethering to known potent FFA1 agonists. This led to the development of 4, a high affinity FFA1 tracer with favorable and polarity-dependent fluorescent properties. A close to ideal overlap between the emission spectrum of the NanoLuciferase receptor tag and the excitation spectrum of 4 enabled the establishment of a homogeneous BRET-based binding assay suitable for both detailed kinetic studies and high throughput competition binding studies. Using 4 as a tracer demonstrated that the compound acts fully competitively with selected synthetic agonists but not with lauric acid and allowed for the characterization of binding affinities of a diverse selection of known FFA1 agonists, indicating that 4 will be a valuable tool for future studies at FFA1. PMID:27074625

  12. Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

    SciTech Connect

    Yonezawa, Tomo Haga, Satoshi; Kobayashi, Yosuke; Katoh, Kazuo; Obara, Yoshiaki

    2008-03-21

    GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.

  13. Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle.

    PubMed

    Mizuta, Kentaro; Zhang, Yi; Mizuta, Fumiko; Hoshijima, Hiroshi; Shiga, Toshiya; Masaki, Eiji; Emala, Charles W

    2015-11-01

    Obesity is one of the major risk factors for asthma. Previous studies have demonstrated that free fatty acid levels are elevated in the plasma of obese individuals. Medium- and long-chain free fatty acids act as endogenous ligands for the free fatty acid receptors FFAR1/GPR40 and FFAR4/GPR120, which couple to Gq proteins. We investigated whether FFAR1 and FFAR4 are expressed on airway smooth muscle and whether they activate Gq-coupled signaling and modulate airway smooth muscle tone. We detected the protein expression of FFAR1 and FFAR4 in freshly dissected native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by immunoblotting and immunohistochemistry. The long-chain free fatty acids (oleic acid and linoleic acid) and GW9508 (FFAR1/FFAR4 dual agonist) dose-dependently stimulated transient intracellular Ca(2+) concentration ([Ca(2+)]i) increases and inositol phosphate synthesis in HASM cells. Downregulation of FFAR1 or FFAR4 in HASM cells by small interfering RNA led to a significant inhibition of the long-chain free fatty acids-induced transient [Ca(2+)]i increases. Oleic acid, linoleic acid, or GW9508 stimulated stress fiber formation in HASM cells, potentiated acetylcholine-contracted guinea pig tracheal rings, and attenuated the relaxant effect of isoproterenol after an acetylcholine-induced contraction. In contrast, TUG-891 (FFAR4 agonist) did not induce the stress fiber formation or potentiate acetylcholine-induced contraction. These results suggest that FFAR1 is the functionally dominant free fatty acid receptor in both human and guinea pig airway smooth muscle. The free fatty acid sensors expressed on airway smooth muscle could be an important modulator of airway smooth muscle tone.

  14. Diets Containing α-Linolenic (ω3) or Oleic (ω9) Fatty Acids Rescues Obese Mice From Insulin Resistance.

    PubMed

    Oliveira, V; Marinho, R; Vitorino, D; Santos, G A; Moraes, J C; Dragano, N; Sartori-Cintra, A; Pereira, L; Catharino, R R; da Silva, A S R; Ropelle, E R; Pauli, J R; De Souza, C T; Velloso, L A; Cintra, D E

    2015-11-01

    Subclinical systemic inflammation is a hallmark of obesity and insulin resistance. The results obtained from a number of experimental studies suggest that targeting different components of the inflammatory machinery may result in the improvement of the metabolic phenotype. Unsaturated fatty acids exert antiinflammatory activity through several distinct mechanisms. Here, we tested the capacity of ω3 and ω9 fatty acids, directly from their food matrix, to exert antiinflammatory activity through the G protein-coupled receptor (GPR)120 and GPR40 pathways. GPR120 was activated in liver, skeletal muscle, and adipose tissues, reverting inflammation and insulin resistance in obese mice. Part of this action was also mediated by GPR40 on muscle, as a novel mechanism described. Pair-feeding and immunoneutralization experiments reinforced the pivotal role of GPR120 as a mediator in the response to the nutrients. The improvement in insulin sensitivity in the high-fat substituted diets was associated with a marked reduction in tissue inflammation, decreased macrophage infiltration, and increased IL-10 levels. Furthermore, improved glucose homeostasis was accompanied by the reduced expression of hepatic gluconeogenic enzymes and reduced body mass. Thus, our data indicate that GPR120 and GPR40 play a critical role as mediators of the beneficial effects of dietary unsaturated fatty acids in the context of obesity-induced insulin resistance. PMID:26280128

  15. [Elucidation of mechanisms underlying docosahexaenoic acid-induced antinociception].

    PubMed

    Nishinaka, Takashi; Matsumoto, Kengo; Nakamoto, Kazuo; Anbo, Akihiro; Mankura, Mitsumasa; Koyama, Yutaka; Tokuyama, Shogo

    2013-01-01

    Docosahexaenoic acid (DHA), a predominant of n-3 polyunsaturated fatty acids (n-3 PUFA), has numerous beneficial physiological effects, including neuroprotection and cardiovascular protection. Recently, a possible involvement of n-3 PUFA in pain control has gathered considerable attention because numerous studies have reported a regulatory role of n-3 PUFAs. However, the mechanisms underlying how DHA exerts antinociceptive effect remain unknown. Here, we performed elucidation of mechanisms underlying DHA-induced antinociception. DHA administration dose-dependently exerted an antinociceptive effect. This effect was abolished by pretreated with the β-funaltrexamine (β-FNA), a μ-opioid receptor antagonist, and the nartrindole (NTI), a δ-opioid receptor antagonist, but not by the nor-binaltorphimine (nor-BNI), a κ-opioid receptor antagonist. In the radioligand binding assay, DHA itself did not have the affinity for μ-, δ- and κ- opioid receptor. Furthermore, the pretreatment of anti β-endorphin antiserum inhibited DHA-induced antinociception. The plasma levels of β-endorphin increased 30 min after DHA administration. The β-endorphin immunoreactivity in the brain increased at 30 min after DHA treatment. Expression of GPR40 protein was widely observed in the brain as well as the spinal cord. The intracerebroventricular but not intrathecal injection of DHA and GW9508, a GPR40/GPR120 agonist, significantly reduced formalin-induced pain behavior. The β-endorphin immunoreactivity in the brain increased at 10 and 20 min after intracerebroventricular injection of DHA and GW9508. These findings suggest that DHA-induced antinociception via β-endorphin release may be mediated through GPR40 signaling in the supraspinal area. PMID:23649389

  16. Design, synthesis and Structure-activity relationship studies of new thiazole-based free fatty acid receptor 1 agonists for the treatment of type 2 diabetes.

    PubMed

    Li, Zheng; Qiu, Qianqian; Xu, Xue; Wang, Xuekun; Jiao, Lei; Su, Xin; Pan, Miaobo; Huang, Wenlong; Qian, Hai

    2016-05-01

    The free fatty acid receptor 1 (FFA1/GPR40) has attracted interest as a novel target for the treatment of type 2 diabetes. Several series of FFA1 agonists including TAK-875, the most advanced compound terminated in phase III studies due to concerns about liver toxicity, have been hampered by relatively high molecular weight and lipophilicity. Aiming to develop potent FFA1 agonists with low risk of liver toxicity by decreasing the lipophilicity, the middle phenyl of TAK-875 was replaced by 11 polar five-membered heteroaromatics. Subsequently, systematic exploration of SAR and application of molecular modeling, leads to the identification of compound 44, which was an excellent FFA1 agonist with robustly hypoglycemic effect both in normal and type 2 diabetic mice, low risks of hypoglycemia and liver toxicity even at the twice molar dose of TAK-875. Meanwhile, two important findings were noted. First, the methyl group in our thiazole series occupied a small hydrophobic subpocket which had no interactions with TAK-875. Furthermore, the agonistic activity revealed a good correlation with the dihedral angle between thiazole core and the terminal benzene ring. These results promote the understanding of ligand-binding pocket and might help to design more promising FFA1 agonists. PMID:26945112

  17. Fasiglifam/TAK-875, a Selective GPR40 Agonist, Improves Hyperglycemia in Rats Unresponsive to Sulfonylureas and Acts Additively with Sulfonylureas.

    PubMed

    Ito, Ryo; Tsujihata, Yoshiyuki; Suzuki, Masami; Miyawaki, Kazumasa; Matsuda, Kae; Takeuchi, Koji

    2016-04-01

    Sulfonylureas (SUs) are widely used insulin secretagogues, but they have adverse effects including hypoglycemia and secondary failure. Fasiglifam/TAK-875, a selective GPR40 agonist, enhances glucose-stimulated insulin secretion and improves hyperglycemia. In the present study, we compared the in vivo glucose-lowering effects of fasiglifam with SUs. The risk of secondary failure of fasiglifam and the efficacy in rats desensitized to SUs were also evaluated. Moreover, we assessed whether fasiglifam was effective when combined with SUs. In diabetic neonatally streptozotocin-induced rats 1.5 days after birth (N-STZ-1.5), oral administrations of fasiglifam (3-30 mg/kg) dose dependently improved glucose tolerance; the effect was greater than that of glibenclamide at maximal effective doses (glucose AUC: fasiglifam, -37.6%; glibenclamide, -12.3%). Although the glucose-lowering effects of glibenclamide (10 mg/kg/day) were completely diminished in N-STZ-1.5 rats after 4 weeks of treatment, effects were maintained in rats receiving fasiglifam (10 mg/kg/day), even after 15 weeks. Fasiglifam (3-10 mg/kg) was still effective in two models desensitized to SUs: 15-week glibenclamide-treated N-STZ-1.5 rats and aged Zucker diabetic fatty (ZDF) rats. Acute administration of fasiglifam (3 mg/kg) and glimepiride (10 mg/kg) in combination additively decreased glucose AUC (fasiglifam, -25.3%; glimepiride, -20.0%; combination, -43.1%). Although glimepiride (10 mg/kg) decreased plasma glucose below normal in nonfasted control rats, fasiglifam (3 mg/kg) maintained normoglycemia, and no further exaggeration of hypoglycemia was observed with combination treatment. These results indicate that GPR40 agonists could be more effective and durable than SUs. Our results also provide new insights into GPR40 pharmacology and rationale for the use of GPR40 agonists in diabetic patients with SU failure. PMID:26813930

  18. Interactions of methoxyacetic acid with androgen receptor

    SciTech Connect

    Bagchi, Gargi; Hurst, Christopher H.; Waxman, David J.

    2009-07-15

    Endocrine disruptive compounds (EDC) alter hormone-stimulated, nuclear receptor-dependent physiological and developmental processes by a variety of mechanisms. One recently identified mode of endocrine disruption is through hormone sensitization, where the EDC modulates intracellular signaling pathways that control nuclear receptor function, thereby regulating receptor transcriptional activity indirectly. Methoxyacetic acid (MAA), the primary, active metabolite of the industrial solvent ethylene glycol monomethyl ether and a testicular toxicant, belongs to this EDC class. Modulation of nuclear receptor activity by MAA could contribute to the testicular toxicity associated with MAA exposure. In the present study, we evaluated the impact of MAA on the transcriptional activity of several nuclear receptors including the androgen receptor (AR), which plays a pivotal role in the development and maturation of spermatocytes. AR transcriptional activity is shown to be increased by MAA through a tyrosine kinase signaling pathway that involves PI3-kinase. In a combinatorial setting with AR antagonists, MAA potentiated the AR response without significantly altering the EC{sub 50} for androgen responsiveness, partially alleviating the antagonistic effect of the anti-androgens. Finally, MAA treatment of TM3 mouse testicular Leydig cells markedly increased the expression of Cyp17a1 and Shbg while suppressing Igfbp3 expression by {approx} 90%. Deregulation of these genes may alter androgen synthesis and action in a manner that contributes to MAA-induced testicular toxicity.

  19. n-3 Fatty Acids Induce Neurogenesis of Predominantly POMC-Expressing Cells in the Hypothalamus.

    PubMed

    Nascimento, Lucas F R; Souza, Gabriela F P; Morari, Joseane; Barbosa, Guilherme O; Solon, Carina; Moura, Rodrigo F; Victório, Sheila C; Ignácio-Souza, Letícia M; Razolli, Daniela S; Carvalho, Hernandes F; Velloso, Lício A

    2016-03-01

    Apoptosis of hypothalamic neurons is believed to play an important role in the development and perpetuation of obesity. Similar to the hippocampus, the hypothalamus presents constitutive and stimulated neurogenesis, suggesting that obesity-associated hypothalamic dysfunction can be repaired. Here, we explored the hypothesis that n-3 polyunsaturated fatty acids (PUFAs) induce hypothalamic neurogenesis. Both in the diet and injected directly into the hypothalamus, PUFAs were capable of increasing hypothalamic neurogenesis to levels similar or superior to the effect of brain-derived neurotrophic factor (BDNF). Most of the neurogenic activity induced by PUFAs resulted in increased numbers of proopiomelanocortin but not NPY neurons and was accompanied by increased expression of BDNF and G-protein-coupled receptor 40 (GPR40). The inhibition of GPR40 was capable of reducing the neurogenic effect of a PUFA, while the inhibition of BDNF resulted in the reduction of global hypothalamic cell. Thus, PUFAs emerge as a potential dietary approach to correct obesity-associated hypothalamic neuronal loss.

  20. G protein-coupled receptors in regulation of body weight.

    PubMed

    Schiöth, Helgi B

    2006-06-01

    In this issue of CNS & Neurological Disorders-Drug Targets, we focus on G protein-coupled receptors (GPCRs) that are involved in regulating body weight. In six reviews, the melanocortins system (including MC4 and MC3 receptors, Agrp, MSH), the NPY receptors (including NPY-Y1, NPY-Y2, and NPY-Y5, PYY3-36), the cannabinoid system (including the development of rimonabant), the ghrelin (GHS, growth hormone secretagogue) system, the monoamine GPCRs (including serotonin, adrenergic and histamine receptors), orexin (hypocretin) system and the galanin receptors are covered. In this overview, an introduction to the GPCRs and the field of central regulation of food intake is provided together with brief mentioning of some other GPCRs that are also implicated in regulation of body weight, such as the melanin-concentrating hormone (MCH), neuromedin U, prolactin-releasing peptide (PrRP), bombesin, cholecystokinin (CCK), Glucagon-like peptide-1 (GLP-1) (and oxyntomodulin), neuropeptide B (NPB) and neuropeptide W (NPW), opioids peptides, free fatty acid (FFA) receptors (GPR40, GPR41). In total over 40 GPCRs are listed that have been implicated to affect regulation of body weight.

  1. Pharmacology of bile acid receptors: Evolution of bile acids from simple detergents to complex signaling molecules.

    PubMed

    Copple, Bryan L; Li, Tiangang

    2016-02-01

    For many years, bile acids were thought to only function as detergents which solubilize fats and facilitate the uptake of fat-soluble vitamins in the intestine. Many early observations; however, demonstrated that bile acids regulate more complex processes, such as bile acids synthesis and immune cell function through activation of signal transduction pathways. These studies were the first to suggest that receptors may exist for bile acids. Ultimately, seminal studies by many investigators led to the discovery of several bile acid-activated receptors including the farnesoid X receptor, the vitamin D receptor, the pregnane X receptor, TGR5, α5 β1 integrin, and sphingosine-1-phosphate receptor 2. Several of these receptors are expressed outside of the gastrointestinal system, indicating that bile acids may have diverse functions throughout the body. Characterization of the functions of these receptors over the last two decades has identified many important roles for these receptors in regulation of bile acid synthesis, transport, and detoxification; regulation of glucose utilization; regulation of fatty acid synthesis and oxidation; regulation of immune cell function; regulation of energy expenditure; and regulation of neural processes such as gastric motility. Through these many functions, bile acids regulate many aspects of digestion ranging from uptake of essential vitamins to proper utilization of nutrients. Accordingly, within a short time period, bile acids moved beyond simple detergents and into the realm of complex signaling molecules. Because of the important processes that bile acids regulate through activation of receptors, drugs that target these receptors are under development for the treatment of several diseases, including cholestatic liver disease and metabolic syndrome. In this review, we will describe the various bile acid receptors, the signal transduction pathways activated by these receptors, and briefly discuss the physiological processes that

  2. Anatomical and pharmacological characterization of excitatory amino acid receptors

    SciTech Connect

    Monaghan, D.T.

    1985-01-01

    The majority of the excitatory neurotransmission in the vertebrate Central Nervous System is thought to be mediated by acidic amino acid neurotransmitters. However, relatively little is known about the excitatory amino acid receptors and their distribution within the CNS. By analyzing radioligand binding to purified synaptic plasma membranes and to thin tissue sections processed for autoradiography, multiple distinct binding sites were found. These binding sites exhibited the pharmacological properties indicative of the excitatory amino acid receptors, which had been identified by electrophysiological techniques. Specifically, L-(/sup 3/H)-glutamate and D-(/sup 3/H)-amino-5-phosphonopentanoate appear to label N-methyl-D-aspartate receptors, L-(/sup 3/H)-glutamate and (/sup 3/H)-kainic acid appear to label kainic acid receptors, and L-(/sup 3/H)-glutamate and (/sup 3/H)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate appear to label quisqualate receptors. Together, these results confirm the three receptor scheme proposed for excitatory amino acid neurotransmission. These results also show that these transmitter-receptor systems are differentially distributed in the brain, and that the total distribution is consistent with that found by other markers for excitatory amino acid-using neurons.

  3. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish. PMID:25732591

  4. Recognition of Legionella pneumophila nucleic acids by innate immune receptors.

    PubMed

    Cunha, Larissa D; Zamboni, Dario S

    2014-12-01

    Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection.

  5. Receptor-level interrelationships of amino acids and the adequate amino acid type hormones in Tetrahymena: a receptor evolution model.

    PubMed

    Csaba, G; Darvas, Z

    1986-01-01

    Histidine stimulates the phagocytosis of Tetrahymena to the same extent as histamine, and also stimulates its division, which histamine does not. Tyrosine and diiodotyrosine equally stimulate the growth of the Tetrahymena. Both amino acids inhibit the characteristic influence of the adequate amino acid hormone when added to Tetrahymena culture 72 h in advance of it. Primary interaction with diiodotyrosine and tyrosine notably increases the cellular growth rate. Histamine has a similar, although less notable effect than histidine. In the light of these experimental observations there is reason to postulate that the receptors of the amino acid hormones have developed from amino acid receptors.

  6. A thermodynamic switch modulates abscisic acid receptor sensitivity

    PubMed Central

    Dupeux, Florine; Santiago, Julia; Betz, Katja; Twycross, Jamie; Park, Sang-Youl; Rodriguez, Lesia; Gonzalez-Guzman, Miguel; Jensen, Malene Ringkjøbing; Krasnogor, Natalio; Blackledge, Martin; Holdsworth, Michael; Cutler, Sean R; Rodriguez, Pedro L; Márquez, José Antonio

    2011-01-01

    Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR)/PYR1-like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracellular receptors promotes the formation of stable complexes with certain protein phosphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest plant hormone receptor family known; however, it is unclear what functional differentiation exists among receptors. Here, we identify two distinct classes of receptors, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms. Moreover, we find a residue in PYR1, H60, that is variable between family members and plays a key role in determining oligomeric state. In silico modelling of the ABA activation pathway reveals that monomeric receptors have a competitive advantage for binding to ABA and PP2Cs. This work illustrates how receptor oligomerization can modulate hormonal responses and more generally, the sensitivity of a ligand-dependent signalling system. PMID:21847091

  7. Bile acid receptors and nonalcoholic fatty liver disease

    PubMed Central

    Yuan, Liyun; Bambha, Kiran

    2015-01-01

    With the high prevalence of obesity, diabetes, and other features of the metabolic syndrome in United States, nonalcoholic fatty liver disease (NAFLD) has inevitably become a very prevalent chronic liver disease and is now emerging as one of the leading indications for liver transplantation. Insulin resistance and derangement of lipid metabolism, accompanied by activation of the pro-inflammatory response and fibrogenesis, are essential pathways in the development of the more clinically significant form of NAFLD, known as nonalcoholic steatohepatitis (NASH). Recent advances in the functional characterization of bile acid receptors, such as farnesoid X receptor (FXR) and transmembrane G protein-coupled receptor (TGR) 5, have provided further insight in the pathophysiology of NASH and have led to the development of potential therapeutic targets for NAFLD and NASH. Beyond maintaining bile acid metabolism, FXR and TGR5 also regulate lipid metabolism, maintain glucose homeostasis, increase energy expenditure, and ameliorate hepatic inflammation. These intriguing features have been exploited to develop bile acid analogues to target pathways in NAFLD and NASH pathogenesis. This review provides a brief overview of the pathogenesis of NAFLD and NASH, and then delves into the biological functions of bile acid receptors, particularly with respect to NASH pathogenesis, with a description of the associated experimental data, and, finally, we discuss the prospects of bile acid analogues in the treatment of NAFLD and NASH. PMID:26668692

  8. Characterization of the hydroxycarboxylic acid receptor 2 in cats.

    PubMed

    Graff, E C; Norris, O C; Sandey, M; Kemppainen, R J; Judd, R L

    2015-10-01

    The hydroxycarboxylic acid receptor 2 (HCA2) belongs to a family of nutrient-sensing receptors that bind β-hydroxybutyrate, an alternative fuel source produced during a negative energy balance. The HCA2 receptor has not been identified or characterized in cats. Therefore, the following were the objectives of this study: (1) identify the feline HCA2 receptor protein sequence and compare against known human and rodent sequences, (2) determine tissue distribution and relative expression in lean, healthy cats, and (3) demonstrate in vitro functionality in feline adipose tissue. Tissues (n = 6) and primary adipocytes (n = 4) were collected from lean, healthy, female cats. The published genomic sequence for cats was used to design primers for polymerase chain reaction isolation of HCA2. Relative tissue distribution was evaluated using reverse transcriptase-polymerase chain reaction with RNA isolated from 9 different tissues (spleen, pancreas, lymph node, jejunum, kidney, liver, heart, and subcutaneous and abdominal adipose tissue). Receptor function was evaluated in primary feline adipocyte culture, and changes were compared with basal lipolysis. The in silico predicted feline HCA2 protein sequence exhibited 83.1% and 86.5% amino acid similarity to human and mouse sequences, respectively. The feline HCA2 receptor is predominantly expressed in adipose tissue and spleen. Exposure of feline adipocytes to niacin, a pharmacologic ligand of HCA2, inhibited lipolysis to a similar degree as insulin, a potent lipolytic inhibitor. In conclusion, the feline HCA2 receptor is similar to human and murine receptors in sequence, distribution, and functionality. By gaining a better understanding of the HCA2 receptor in cats, we will be able to better manage feline patients.

  9. Carbobenzoxy amino acids: Structural requirements for cholecystokinin receptor antagonist activity

    SciTech Connect

    Maton, P.N.; Sutliff, V.E.; Jensen, R.T.; Gardner, J.D.

    1985-04-01

    The authors used dispersed acini prepared from guinea pig pancreas to examine 28 carbobenzoxy (CBZ) amino acids for their abilities to function as cholecystokinin receptor antagonists. All amino acid derivatives tested, except for CBZ-alanine, CBZ-glycine, and N alpha-CBZ- lysine, were able to inhibit the stimulation of amylase secretion caused by the C-terminal octapeptide of cholecystokinin. In general, there was a good correlation between the ability of a carbobenzoxy amino acid to inhibit stimulated amylase secretion and the ability of the amino acid derivative to inhibit binding of /sup 125/I-cholecystokinin. The inhibition of cholecystokinin-stimulated amylase secretion was competitive, fully reversible, and specific for those secretagogues that interact with the cholecystokinin receptor. The potencies with which the various carbobenzoxy amino acids inhibited the action of cholecystokinin varied 100-fold and CBZ-cystine was the most potent cholecystokinin receptor antagonist. This variation in potency was primarily but not exclusively a function of the hydrophobicity of the amino acid side chain.

  10. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    PubMed

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  11. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  12. Bile acid nuclear receptor FXR and digestive system diseases

    PubMed Central

    Ding, Lili; Yang, Li; Wang, Zhengtao; Huang, Wendong

    2015-01-01

    Bile acids (BAs) are not only digestive surfactants but also important cell signaling molecules, which stimulate several signaling pathways to regulate some important biological processes. The bile-acid-activated nuclear receptor, farnesoid X receptor (FXR), plays a pivotal role in regulating bile acid, lipid and glucose homeostasis as well as in regulating the inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. As expected, FXR is involved in the pathophysiology of a wide range of diseases of gastrointestinal tract, including inflammatory bowel disease, colorectal cancer and type 2 diabetes. In this review, we discuss current knowledge of the roles of FXR in physiology of the digestive system and the related diseases. Better understanding of the roles of FXR in digestive system will accelerate the development of FXR ligands/modulators for the treatment of digestive system diseases. PMID:26579439

  13. Bile acid nuclear receptor FXR and digestive system diseases.

    PubMed

    Ding, Lili; Yang, Li; Wang, Zhengtao; Huang, Wendong

    2015-03-01

    Bile acids (BAs) are not only digestive surfactants but also important cell signaling molecules, which stimulate several signaling pathways to regulate some important biological processes. The bile-acid-activated nuclear receptor, farnesoid X receptor (FXR), plays a pivotal role in regulating bile acid, lipid and glucose homeostasis as well as in regulating the inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. As expected, FXR is involved in the pathophysiology of a wide range of diseases of gastrointestinal tract, including inflammatory bowel disease, colorectal cancer and type 2 diabetes. In this review, we discuss current knowledge of the roles of FXR in physiology of the digestive system and the related diseases. Better understanding of the roles of FXR in digestive system will accelerate the development of FXR ligands/modulators for the treatment of digestive system diseases. PMID:26579439

  14. Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics

    PubMed Central

    Solt, Laura A.; Griffin, Patrick R.; Burris, Thomas P.

    2016-01-01

    Purpose of review In the late 1980s, the cloning of several nuclear receptors led to the intense search and isolation of new members of this superfamily. Despite their identification, many of these receptors were dubbed ‘orphan’ receptors, as their physiological ligands remained unknown. Recent reports have presented evidence for one family of orphan receptors, the retinoic acid receptor-related orphan receptors (RORs), in several pathologies, including osteoporosis, several autoimmune diseases, asthma, cancer, diabetes and obesity. The present review summarizes the studies identifying ligands for the RORs and evaluates their role as targets for potential therapeutics. Recent findings Significant progress was made in the initial identification of ligands for the RORs when X-ray crystallographic studies identified several molecules within the ligand-binding pockets of RORα and RORβ. Recently, we identified endogenous and synthetic ligands for RORα and RORγ, thereby solidifying their function as ligand-dependent transcription factors. Summary Recent studies have established roles for the RORs in physiological development and the advent of disease. Identification of ligands for the RORs, both endogenous and synthetic, has established these receptors as attractive new therapeutic targets for the treatment of ROR-related diseases. PMID:20463469

  15. Seizure control by decanoic acid through direct AMPA receptor inhibition.

    PubMed

    Chang, Pishan; Augustin, Katrin; Boddum, Kim; Williams, Sophie; Sun, Min; Terschak, John A; Hardege, Jörg D; Chen, Philip E; Walker, Matthew C; Williams, Robin S B

    2016-02-01

    The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones β-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet. PMID:26608744

  16. Lysophosphatidic acid receptor (LPAR) modulators: The current pharmacological toolbox.

    PubMed

    Llona-Minguez, Sabin; Ghassemian, Artin; Helleday, Thomas

    2015-04-01

    Lysophosphatidic acids (LPA) are key lipid-signalling molecules that regulate a remarkably diverse set of cellular events, such as motility, chemotaxis, cell cycle progression, viability, and wound healing. The physiological and pathophysiological consequences of LPA signalling are evident and misregulation of LPA signalling can lead to pathologies like cancer, atherosclerosis, ischaemia, and fibrosis. LPA exerts its biological actions mainly through several types of G protein-coupled receptors, some of which display opposing or redundant effects. For this reason, selective LPA receptor small-molecule ligands can shine light on LPA biology and present an exciting opportunity for drug discovery endeavours. This review provides insights into the detailed chemical nature and pharmacological profile of the small-molecules thus far developed as LPA receptor modulators, as well as information on the preparation of key pharmaceuticals. This summary will facilitate future research efforts and nurture collaboration between chemists and biologists working in this emerging field. PMID:25704399

  17. Retinoic acid receptor alpha (RAralpha) Mutations in Human Leukemia.

    PubMed

    Parrado, A; Chomienne, C; Padua, R A

    2000-10-01

    The retinoic acid receptor alpha (RARalpha) plays a central role in the biology of the myeloid cellular compartment. Chromosomal translocations involving the RARalpha locus probably represent the malignant initiating events in acute promyelocytic leukemia (APL). Recent studies that identify novel interactions between RARalpha and the nuclear receptor co-activators and co-repressors, new functions of the oncogenic RARalpha fusion proteins and their catabolism in retinoic acid-induced differentiation, and the availability of new transgenic mice models have provided important insights into our understanding of the mechanisms by which mutant forms of RARalpha can be implicated in the development of leukemia. Novel alterations of the RARalpha gene identified in hematopoietic malignant disorders other than APL, such as myelodysplastic syndromes, non-APL acute myeloid leukemias and B-chronic lymphocytic leukemias, suggest that disruption of the RARalpha gene might predispose to myeloid and lymphoid disorders.

  18. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    SciTech Connect

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  19. A third human retinoic acid receptor, hRAR-. gamma

    SciTech Connect

    Krust, A.; Kastner, Ph.; Petkovich, M.; Zelent, A.; Chambon, P. )

    1989-07-01

    Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. The authors have previously characterized two human RAR (hRAR-{alpha} and hRAR-{beta}) cDNAs and have recently cloned their murine cognates (mRAR-{alpha} and mRAR-{beta}) together with a third RAR (mRAR-{gamma}) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-{gamma} cDNA was used here to clone its human counterpart (hRAR-{gamma}) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, they demonstrate that hRAR-{gamma} cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either {alpha}, {beta}, or {gamma}) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-{alpha}, -{beta}, and -{gamma} may perform specific functions. They show also that hRAR-{gamma} RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-{gamma} mediates some of the retinoid effects in this tissue.

  20. Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor

    PubMed Central

    Arnberg, Niklas; Edlund, Karin; Kidd, Alistair H.; Wadell, Göran

    2000-01-01

    Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2. PMID:10590089

  1. Cholestenoic acids regulate motor neuron survival via liver X receptors

    PubMed Central

    Theofilopoulos, Spyridon; Griffiths, William J.; Crick, Peter J.; Yang, Shanzheng; Meljon, Anna; Ogundare, Michael; Kitambi, Satish Srinivas; Lockhart, Andrew; Tuschl, Karin; Clayton, Peter T.; Morris, Andrew A.; Martinez, Adelaida; Reddy, M. Ashwin; Martinuzzi, Andrea; Bassi, Maria T.; Honda, Akira; Mizuochi, Tatsuki; Kimura, Akihiko; Nittono, Hiroshi; De Michele, Giuseppe; Carbone, Rosa; Criscuolo, Chiara; Yau, Joyce L.; Seckl, Jonathan R.; Schüle, Rebecca; Schöls, Ludger; Sailer, Andreas W.; Kuhle, Jens; Fraidakis, Matthew J.; Gustafsson, Jan-Åke; Steffensen, Knut R.; Björkhem, Ingemar; Ernfors, Patrik; Sjövall, Jan; Arenas, Ernest; Wang, Yuqin

    2014-01-01

    Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death. PMID:25271621

  2. Development of novel silicon-containing inverse agonists of retinoic acid receptor-related orphan receptors.

    PubMed

    Toyama, Hirozumi; Nakamura, Masaharu; Nakamura, Masahiko; Matsumoto, Yotaro; Nakagomi, Madoka; Hashimoto, Yuichi

    2014-03-15

    Retinoic acid receptor (RAR)-related orphan receptors (RORs) regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm and immune function. The RAR agonist: all-trans retinoic acid was reported to be an RORβ inverse agonist, but no information is available regarding ROR activity of its synthetic analogue Am580. Therefore, we screened Am580 and some related tetramethyltetrahydronaphthalene derivatives and carried out structural development studies, including substitution of carbon atoms with silicon, with the aim of creating a potent ROR transcriptional inhibitor. The phenyl amide disila compound 22 showed the most potent ROR-inhibitory activity among the compounds examined. Its activity towards RORα, RORβ and RORγ was increased compared to that of Am580. The IC₅₀ values for RORα, RORβ and RORγ are 1.3, >10 and 4.5 μM, respectively.

  3. Receptor for protons: First observations on Acid Sensing Ion Channels.

    PubMed

    Krishtal, Oleg

    2015-07-01

    The history of ASICs began in 1980 with unexpected observation. The concept of highly selective Na(+) current gated by specific receptors for protons was not easily accepted. It took 16 years to get these receptor/channels cloned and start a new stage in their investigation. "The receptor for protons" became ASIC comprising under this name a family of receptor/channels ubiquitous for mammalian nervous system, both peripheral and central. The role of ASICs as putative nociceptors was suggested almost immediately after their discovery. This role subsequently was proven in many forms of pain-related phenomena. Many other functions of ASICs have been also found or primed for speculations both in physiology and in disease. Despite the width of field and strength of efforts, numerous basic questions are to be answered before we understand how the local changes in pH in the nervous tissue transform into electric and messenger signaling via ASICs as transducers. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.

  4. Bridging cell surface receptor with nuclear receptors in control of bile acid homeostasis

    PubMed Central

    Li, Shuangwei; Ni, Andrew; Feng, Gen-sheng

    2015-01-01

    Bile acids (BAs) are traditionally considered as “physiological detergents” for emulsifying hydrophobic lipids and vitamins due to their amphipathic nature. But accumulating clinical and experimental evidence shows an association between disrupted BA homeostasis and various liver disease conditions including hepatitis infection, diabetes and cancer. Consequently, BA homeostasis regulation has become a field of heavy interest and investigation. After identification of the Farnesoid X Receptor (FXR) as an endogenous receptor for BAs, several nuclear receptors (SHP, HNF4α, and LRH-1) were also found to be important in regulation of BA homeostasis. Some post-translational modifications of these nuclear receptors have been demonstrated, but their physiological significance is still elusive. Gut secrets FGF15/19 that can activate hepatic FGFR4 and its downstream signaling cascade, leading to repressed hepatic BA biosynthesis. However, the link between the activated kinases and these nuclear receptors is not fully elucidated. Here, we review the recent literature on signal crosstalk in BA homeostasis. PMID:25500873

  5. Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer

    PubMed Central

    Johansson, Henrik J.; Sanchez, Betzabe C.; Mundt, Filip; Forshed, Jenny; Kovacs, Aniko; Panizza, Elena; Hultin-Rosenberg, Lina; Lundgren, Bo; Martens, Ulf; Máthé, Gyöngyvér; Yakhini, Zohar; Helou, Khalil; Krawiec, Kamilla; Kanter, Lena; Hjerpe, Anders; Stål, Olle; Linderholm, Barbro K.; Lehtiö, Janne

    2013-01-01

    About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen. PMID:23868472

  6. Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis

    PubMed Central

    Lin, Kuan-Hung; Ho, Ya-Hsuan; Chiang, Jui-Chung; Li, Meng-Wei; Lin, Shi-Hung; Chen, Wei-Min; Chiang, Chi-Ling; Lin, Yu-Nung; Yang, Ya-Jan; Chen, Chiung-Nien; Lu, Jenher; Huang, Chang-Jen; Tigyi, Gabor; Yao, Chao-Ling; Lee, Hsinyu

    2016-01-01

    Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34+ human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis. PMID:27244685

  7. Retinoic acid receptors: from molecular mechanisms to cancer therapy.

    PubMed

    di Masi, Alessandra; Leboffe, Loris; De Marinis, Elisabetta; Pagano, Francesca; Cicconi, Laura; Rochette-Egly, Cécile; Lo-Coco, Francesco; Ascenzi, Paolo; Nervi, Clara

    2015-02-01

    Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported. PMID:25543955

  8. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    PubMed Central

    Wellendorph, P; Bräuner-Osborne, H

    2009-01-01

    Family C of human G-protein-coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two γ-aminobutyric acid type B (GABAB1–2) subunits forming the heterodimeric GABAB receptor, the calcium-sensing receptor, three taste1 receptors (T1R1–3), a promiscuous L-α-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABAB1–2 and T1R2–3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity and the initial steps in receptor activation. PMID:19298394

  9. Leveraging abscisic acid receptors for efficient water use in Arabidopsis.

    PubMed

    Yang, Zhenyu; Liu, Jinghui; Tischer, Stefanie V; Christmann, Alexander; Windisch, Wilhelm; Schnyder, Hans; Grill, Erwin

    2016-06-14

    Plant growth requires the influx of atmospheric CO2 through stomatal pores, and this carbon uptake for photosynthesis is inherently associated with a large efflux of water vapor. Under water deficit, plants reduce transpiration and are able to improve carbon for water exchange leading to higher water use efficiency (WUE). Whether increased WUE can be achieved without trade-offs in plant growth is debated. The signals mediating the WUE response under water deficit are not fully elucidated but involve the phytohormone abscisic acid (ABA). ABA is perceived by a family of related receptors known to mediate acclimation responses and to reduce transpiration. We now show that enhanced stimulation of ABA signaling via distinct ABA receptors can result in plants constitutively growing at high WUE in the model species Arabidopsis WUE was assessed by three independent approaches involving gravimetric analyses, (13)C discrimination studies of shoots and derived cellulose fractions, and by gas exchange measurements of whole plants and individual leaves. Plants expressing the ABA receptors RCAR6/PYL12 combined up to 40% increased WUE with high growth rates, i.e., are water productive. Water productivity was associated with maintenance of net carbon assimilation by compensatory increases of leaf CO2 gradients, thereby sustaining biomass acquisition. Leaf surface temperatures and growth potentials of plants growing under well-watered conditions were found to be reliable indicators for water productivity. The study shows that ABA receptors can be explored to generate more plant biomass per water transpired, which is a prime goal for a more sustainable water use in agriculture. PMID:27247417

  10. Leveraging abscisic acid receptors for efficient water use in Arabidopsis

    PubMed Central

    Yang, Zhenyu; Liu, Jinghui; Tischer, Stefanie V.; Christmann, Alexander; Windisch, Wilhelm; Schnyder, Hans; Grill, Erwin

    2016-01-01

    Plant growth requires the influx of atmospheric CO2 through stomatal pores, and this carbon uptake for photosynthesis is inherently associated with a large efflux of water vapor. Under water deficit, plants reduce transpiration and are able to improve carbon for water exchange leading to higher water use efficiency (WUE). Whether increased WUE can be achieved without trade-offs in plant growth is debated. The signals mediating the WUE response under water deficit are not fully elucidated but involve the phytohormone abscisic acid (ABA). ABA is perceived by a family of related receptors known to mediate acclimation responses and to reduce transpiration. We now show that enhanced stimulation of ABA signaling via distinct ABA receptors can result in plants constitutively growing at high WUE in the model species Arabidopsis. WUE was assessed by three independent approaches involving gravimetric analyses, 13C discrimination studies of shoots and derived cellulose fractions, and by gas exchange measurements of whole plants and individual leaves. Plants expressing the ABA receptors RCAR6/PYL12 combined up to 40% increased WUE with high growth rates, i.e., are water productive. Water productivity was associated with maintenance of net carbon assimilation by compensatory increases of leaf CO2 gradients, thereby sustaining biomass acquisition. Leaf surface temperatures and growth potentials of plants growing under well-watered conditions were found to be reliable indicators for water productivity. The study shows that ABA receptors can be explored to generate more plant biomass per water transpired, which is a prime goal for a more sustainable water use in agriculture. PMID:27247417

  11. Biological roles and therapeutic potential of hydroxy-carboxylic Acid receptors.

    PubMed

    Ahmed, Kashan

    2011-01-01

    In the recent past, deorphanization studies have described intermediates of energy metabolism to activate G protein-coupled receptors and to thereby regulate metabolic functions. GPR81, GPR109A, and GPR109B, formerly known as the nicotinic acid receptor family, are encoded by clustered genes and share a high degree of sequence homology. Recently, hydroxy-carboxylic acids were identified as endogenous ligands of GPR81, GPR109A, and GPR109B, and therefore these receptors have been placed into a novel receptor family of hydroxy-carboxylic acid (HCA) receptors. The HCA(1) receptor (GPR81) is activated by the glycolytic metabolite 2-hydroxy-propionic acid (lactate), the HCA(2) receptor is activated by the ketone body 3-hydroxy-butyric acid, and the HCA(3) receptor (GPR109B) is a receptor for the β-oxidation intermediate 3-hydroxy-octanoic acid. While HCA(1) and HCA(2) receptors are present in most mammalian species, the HCA(3) receptor is exclusively found in humans and higher primates. HCA receptors are expressed in adipose tissue and mediate anti-lipolytic effects in adipocytes through G(i)-type G protein-dependent inhibition of adenylyl cyclase. HCA(2) and HCA(3) inhibit lipolysis during conditions of increased β-oxidation such as prolonged fasting, whereas HCA(1) mediates the anti-lipolytic effects of insulin in the fed state. As HCA(2) is a receptor for the established anti-dyslipidemic drug nicotinic acid, HCA(1) and HCA(3) also represent promising drug targets and several synthetic ligands for HCA receptors have been developed. In this article, we will summarize the deorphanization and pharmacological characterization of HCA receptors. Moreover, we will discuss recent progress in elucidating the physiological and pathophysiological role to further evaluate the therapeutic potential of the HCA receptor family for the treatment of metabolic disease.

  12. Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α.

    PubMed

    Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D; Sanyal, Sabyasachi

    2011-06-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.

  13. A widely used retinoic acid receptor antagonist induces peroxisome proliferator-activated receptor-gamma activity.

    PubMed

    Schupp, Michael; Curtin, Joshua C; Kim, Roy J; Billin, Andrew N; Lazar, Mitchell A

    2007-05-01

    Nuclear receptors (NRs) are transcription factors whose activity is regulated by the binding of small lipophilic ligands, including hormones, vitamins, and metabolites. Pharmacological NR ligands serve as important therapeutic agents; for example, all-trans retinoic acid, an activating ligand for retinoic acid receptor alpha (RARalpha), is used to treat leukemia. Another RARalpha ligand, (E)-S,S-dioxide-4-(2-(7-(heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)-1-propenyl)-benzoic acid (Ro 41-5253), is a potent antagonist that has been a useful and purportedly specific probe of RARalpha function. Here, we report that Ro 41-5253 also activates the peroxisome proliferator-activated receptor gamma (PPARgamma), a master regulator of adipocyte differentiation and target of widely prescribed antidiabetic thiazolidinediones (TZDs). Ro 41-5253 enhanced differentiation of mouse and human preadipocytes and activated PPARgamma target genes in mature adipocytes. Like the TZDs, Ro 41-5253 also down-regulated PPARgamma protein expression in adipocytes. In addition, Ro 41-5253 activated the PPARgamma-ligand binding domain in transiently transfected HEK293T cells. These effects were not prevented by a potent RARalpha agonist or by depleting cells of RARalpha, indicating that PPARgamma activation was not related to RARalpha antagonism. Indeed, Ro 41-5253 was able to compete with TZD ligands for binding to PPARgamma, suggesting that Ro 41-5253 directly affects PPAR activity. These results vividly demonstrate that pharmacological NR ligands may have "off-target" effects on other NRs. Ro 41-5253 is a PPARgamma agonist as well as an RARalpha antagonist whose pleiotropic effects on NRs may signify a unique spectrum of biological responses.

  14. Thyroid hormone receptor can modulate retinoic acid-mediated axis formation in frog embryogenesis.

    PubMed Central

    Banker, D E; Eisenman, R N

    1993-01-01

    Thyroid hormone receptor acts as a hormone-dependent transcriptional transactivator and as a transcriptional repressor in the absence of thyroid hormone. Specifically, thyroid hormone receptor can repress retinoic acid-induced gene expression through interactions with retinoic acid receptor. (Retinoic acid is a potent teratogen in the frog Xenopus laevis, acting at early embryonic stages to interfere with the formation of anterior structures. Endogenous retinoic acid is thought to act in normal anterior-posterior axis formation.) We have previously shown that thyroid hormone receptor RNA (alpha isotype) is expressed and polysome-associated during Xenopus embryogenesis preceding thyroid gland maturation and endogenous thyroid hormone production (D. E. Banker, J. Bigler, and R. N. Eisenman, Mol. Cell. Biol. 11:5079-5089, 1991). To determine whether thyroid hormone receptor might influence the effects of retinoic acid in early frog development, we have examined the results of ectopic thyroid hormone receptor expression on retinoic acid teratogenesis. We demonstrate that microinjections of full-length thyroid hormone receptor RNA protect injected embryos from retinoic acid teratogenesis. DNA binding is apparently essential to this protective function, as truncated thyroid hormone receptors, lacking DNA-binding domains but including hormone-binding and dimerization domains, do not protect from retinoic acid. We have shown that microinjections of these dominant-interfering thyroid hormone receptors, as well as anti-thyroid hormone receptor antibodies, increase retinoic acid teratogenesis in injected embryos, presumably by inactivating endogenous thyroid hormone receptor. This finding suggests that endogenous thyroid hormone receptors may act to limit retinoic acid sensitivity. On the other hand, after thyroid hormone treatment, ectopic thyroid hormone receptor mediates teratogenesis that is indistinguishable from the dorsoanterior deficiencies produced in retinoic acid

  15. FATTY ACIDS MODULATE TOLL-LIKE RECEPTOR 4 ACTIVATION THROUGH REGULATION OF RECEPTOR DIMERIZATION AND RECRUITMENT INTO LIPID RAFTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies (J Biol Chem 2003, 2004) demonstrated that saturated ...

  16. Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.

    PubMed Central

    Elner, Victor M

    2002-01-01

    PURPOSE: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD). METHODS: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipase in situ, in vitro, and after challenge with phagocytic stimuli. Receptor-mediated RPE uptake of fluorescently labeled native, aceto-acetylated, and oxidized LDL was studied in vitro and in vivo. LDL effects on RPE lysosomal enzymes were assessed. Lysosomal enzyme activity was compared in RPE cells from monkeys fed diets rich in fish oil to those from control animals and in cultured RPE cells exposed to sera from these monkeys. RESULTS: RPE acid lipase activity was substantial and comparable to that of mononuclear phagocytes. Acid lipase activity increased significantly following phagocytic challenge with photoreceptor outer segment (POS) membranes. Receptor-mediated RPE uptake of labeled lipoproteins was determined in vitro. Distinctive uptake of labeled lipoproteins occurred in RPE cells and mononuclear phagocytes in vivo. Native LDL enhanced RPE lysosomal enzyme activity. RPE lysosomal enzymes increased significantly in RPE cells from monkeys fed fish oil-rich diets and in cultured RPE cells exposed to their sera. CONCLUSIONS: RPE cells contain substantial acid lipase for efficient metabolism of lipids imbibed by POS phagocytosis and LDL uptake. Diets rich in fish oil-derived omega-3 fatty acids, by enhancing acid lipase, may reduce RPE lipofuscin accumulation, RPE oxidative damage, and the development of ARMD. PMID:12545699

  17. EMBO Retinoids 2011: mechanisms, biology and pathology of signaling by retinoic acid and retinoic acid receptors

    PubMed Central

    McKenna, Neil J.

    2012-01-01

    Retinoic acid (RA) is one of the principal active metabolites of vitamin A (retinol) which mediates a spectrum of critical physiological and developmental processes. Transcriptional regulation by RA is mediated primarily by members of the retinoic acid receptor (RAR) subfamily of the nuclear receptor (NR) superfamily of transcription factors. NRs bind specific genomic DNA sequence motifs and engage coregulators and components of the basal transcription machinery to effect transcriptional regulation at target gene promoters. Disruption of signaling by retinoic acid is thought to underlie the etiology of a number of inflammatory and neoplastic diseases including breast cancer and haematological malignancies. A meeting of international researchers in retinoid signaling was convened in Strasbourg in September 2011 under the auspices of the European Molecular Biology Organization (EMBO). Retinoids 2011 encompassed myriad mechanistic, biological and pathological aspects of these hormones and their cognate receptors, as well as setting these advances in the context of wider current questions on signaling by members of the NR superfamily. PMID:22438793

  18. Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation.

    PubMed

    Yan, Yiqing; Jiang, Wei; Spinetti, Thibaud; Tardivel, Aubry; Castillo, Rosa; Bourquin, Carole; Guarda, Greta; Tian, Zhigang; Tschopp, Jurg; Zhou, Rongbin

    2013-06-27

    Omega-3 fatty acids (ω-3 FAs) have potential anti-inflammatory activity in a variety of inflammatory human diseases, but the mechanisms remain poorly understood. Here we show that stimulation of macrophages with ω-3 FAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other family members, abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1β secretion. In addition, G protein-coupled receptor 120 (GPR120) and GPR40 and their downstream scaffold protein β-arrestin-2 were shown to be involved in inflammasome inhibition induced by ω-3 FAs. Importantly, ω-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced type 2 diabetes model. Our results reveal a mechanism through which ω-3 FAs repress inflammation and prevent inflammation-driven diseases and suggest the potential clinical use of ω-3 FAs in gout, autoinflammatory syndromes, or other NLRP3 inflammasome-driven inflammatory diseases.

  19. Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation.

    PubMed

    Yan, Yiqing; Jiang, Wei; Spinetti, Thibaud; Tardivel, Aubry; Castillo, Rosa; Bourquin, Carole; Guarda, Greta; Tian, Zhigang; Tschopp, Jurg; Zhou, Rongbin

    2013-06-27

    Omega-3 fatty acids (ω-3 FAs) have potential anti-inflammatory activity in a variety of inflammatory human diseases, but the mechanisms remain poorly understood. Here we show that stimulation of macrophages with ω-3 FAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other family members, abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1β secretion. In addition, G protein-coupled receptor 120 (GPR120) and GPR40 and their downstream scaffold protein β-arrestin-2 were shown to be involved in inflammasome inhibition induced by ω-3 FAs. Importantly, ω-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced type 2 diabetes model. Our results reveal a mechanism through which ω-3 FAs repress inflammation and prevent inflammation-driven diseases and suggest the potential clinical use of ω-3 FAs in gout, autoinflammatory syndromes, or other NLRP3 inflammasome-driven inflammatory diseases. PMID:23809162

  20. Medium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory Receptor

    PubMed Central

    Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

    2013-01-01

    G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [35S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease. PMID:23449982

  1. Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

    PubMed

    Marchwicka, Aleksandra; Cebrat, Małgorzata; Łaszkiewicz, Agnieszka; Śnieżewski, Łukasz; Brown, Geoffrey; Marcinkowska, Ewa

    2016-05-01

    Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D. PMID:26969398

  2. Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid.

    PubMed

    Powell, William S; Rokach, Joshua

    2015-04-01

    Arachidonic acid can be oxygenated by a variety of different enzymes, including lipoxygenases, cyclooxygenases, and cytochrome P450s, and can be converted to a complex mixture of oxygenated products as a result of lipid peroxidation. The initial products in these reactions are hydroperoxyeicosatetraenoic acids (HpETEs) and hydroxyeicosatetraenoic acids (HETEs). Oxoeicosatetraenoic acids (oxo-ETEs) can be formed by the actions of various dehydrogenases on HETEs or by dehydration of HpETEs. Although a large number of different HETEs and oxo-ETEs have been identified, this review will focus principally on 5-oxo-ETE, 5S-HETE, 12S-HETE, and 15S-HETE. Other related arachidonic acid metabolites will also be discussed in less detail. 5-Oxo-ETE is synthesized by oxidation of the 5-lipoxygenase product 5S-HETE by the selective enzyme, 5-hydroxyeicosanoid dehydrogenase. It actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, suggesting that it may be important in eosinophilic diseases such as asthma. 5-Oxo-ETE also appears to stimulate tumor cell proliferation and may also be involved in cancer. Highly selective and potent OXE receptor antagonists have recently become available and could help to clarify its pathophysiological role. The 12-lipoxygenase product 12S-HETE acts by the GPR31 receptor and promotes tumor cell proliferation and metastasis and could therefore be a promising target in cancer therapy. It may also be involved as a proinflammatory mediator in diabetes. In contrast, 15S-HETE may have a protective effect in cancer. In addition to GPCRs, higher concentration of HETEs and oxo-ETEs can activate peroxisome proliferator-activated receptors (PPARs) and could potentially regulate a variety of processes by this mechanism. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

  3. Tulane virus recognizes sialic acids as cellular receptors.

    PubMed

    Tan, Ming; Wei, Chao; Huang, Pengwei; Fan, Qiang; Quigley, Christina; Xia, Ming; Fang, Hao; Zhang, Xufu; Zhong, Weiming; Klassen, John S; Jiang, Xi

    2015-01-01

    The recent discovery that human noroviruses (huNoVs) recognize sialic acids (SAs) in addition to histo-blood group antigens (HBGAs) pointed to a new direction in studying virus-host interactions during calicivirus infection. HuNoVs remain difficult to study due to the lack of an effective cell culture model. In this study, we demonstrated that Tulane virus (TV), a cultivable primate calicivirus, also recognizes SAs in addition to the previously known TV-HBGA interactions. Evidence supporting this discovery includes that TV virions bound synthetic sialoglycoconjugates (SGCs) and that treatment of TV permissive LLC-MK2 cells with either neuraminidases or SA-binding lectins inhibited TV infectivity. In addition, we found that Maackia amurensis leukoagglutinin (MAL), a lectin that recognizes the α-2,3 linked SAs, bound LLC-MK2 cells, as well as TV, by which MAL promoted TV infectivity in cell culture. Our findings further highlight TV as a valuable surrogate for huNoVs, particularly in studying virus-host interactions that may involve two host carbohydrate receptors or co-receptors for infection. PMID:26146020

  4. G protein-coupled receptors: signalling and regulation by lipid agonists for improved glucose homoeostasis.

    PubMed

    Moran, Brian M; Flatt, Peter R; McKillop, Aine M

    2016-04-01

    G protein-coupled receptors (GPCRs) play a pivotal role in cell signalling, controlling many processes such as immunity, growth, cellular differentiation, neurological pathways and hormone secretions. Fatty acid agonists are increasingly recognised as having a key role in the regulation of glucose homoeostasis via stimulation of islet and gastrointestinal GPCRs. Downstream cell signalling results in modulation of the biosynthesis, secretion, proliferation and anti-apoptotic pathways of islet and enteroendocrine cells. GPR40 and GPR120 are activated by long-chain fatty acids (>C12) with both receptors coupling to the Gαq subunit that activates the Ca(2+)-dependent pathway. GPR41 and GPR43 are stimulated by short-chain fatty acids (C2-C5), and activation results in binding to Gαi that inhibits the adenylyl cyclase pathway attenuating cAMP production. In addition, GPR43 also couples to the Gαq subunit augmenting intracellular Ca(2+) and activating phospholipase C. GPR55 is specific for cannabinoid endogenous agonists (endocannabinoids) and non-cannabinoid fatty acids, which couples to Gα12/13 and Gαq proteins, leading to enhancing intracellular Ca(2+), extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and Rho kinase. GPR119 is activated by fatty acid ethanolamides and binds to Gαs utilising the adenylate cyclase pathway, which is dependent upon protein kinase A. Current research indicates that GPCR therapies may be approved for clinical use in the near future. This review focuses on the recent advances in preclinical diabetes research in the signalling and regulation of GPCRs on islet and enteroendocrine cells involved in glucose homoeostasis.

  5. Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor.

    PubMed

    Murakami, Masanori; Shiraishi, Akira; Tabata, Kenichi; Fujita, Norihisa

    2008-07-11

    Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y(10) receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y(10) receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca(2+) increases in the CHO cells stably expressing the P2Y(10) fused with a G(16alpha) protein. These Ca(2+) responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y(10) receptor into the P2Y(10)-CHO cells effectively blocked both S1P- and LPA-induced Ca(2+) increases. RT-PCR analysis showed that the mouse P2Y(10) was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y(10) receptor is the first receptor identified as a dual lysophospholipid receptor. PMID:18466763

  6. Cinnabarinic acid, an endogenous metabolite of the kynurenine pathway, activates type 4 metabotropic glutamate receptors.

    PubMed

    Fazio, F; Lionetto, L; Molinaro, G; Bertrand, H O; Acher, F; Ngomba, R T; Notartomaso, S; Curini, M; Rosati, O; Scarselli, P; Di Marco, R; Battaglia, G; Bruno, V; Simmaco, M; Pin, J P; Nicoletti, F; Goudet, C

    2012-05-01

    Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity. PMID:22311707

  7. Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

    SciTech Connect

    Tang, Yuting . E-mail: ytang@prdus.jnj.com; Zhou, Lubing; Gunnet, Joseph W.; Wines, Pamela G.; Cryan, Ellen V.; Demarest, Keith T.

    2006-06-23

    HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A{sub 2} (PLA{sub 2})/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca{sup 2+}-mobilization and enhanced bradykinin-promoted Ca{sup 2+}-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPAR{gamma} agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.

  8. Obeticholic acid, a synthetic bile acid agonist of the farnesoid X receptor, attenuates experimental autoimmune encephalomyelitis

    PubMed Central

    Ho, Peggy P.; Steinman, Lawrence

    2016-01-01

    Bile acids are ligands for the nuclear hormone receptor, farnesoid X receptor (FXR). The bile acid–FXR interaction regulates bile acid synthesis, transport, and cholesterol metabolism. Recently, bile acid–FXR regulation has been reported to play an integral role in both hepatic and intestinal inflammation, and in atherosclerosis. In this study, we found that FXR knockout mice had more disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Obeticholic acid (6α-ethyl-chenodeoxycholic acid, 6-ECDCA), a synthetic FXR agonist, is an orally available drug that is currently in clinical trials for the treatment of inflammatory diseases such as alcoholic hepatitis, nonalcoholic steatohepatitis, and primary biliary cirrhosis. When we treated mice exhibiting established EAE with 6-ECDCA, or the natural FXR ligand chenodeoxycholic acid (CDCA), clinical disease was ameliorated by (i) suppressing lymphocyte activation and proinflammatory cytokine production; (ii) reducing CD4+ T cells and CD19+ B cell populations and their expression of negative checkpoint regulators programmed cell death protein 1 (PD1), programmed death-ligand 1 (PD-L1), and B and T lymphocyte attenuator (BTLA); (iii) increasing CD8+ T cells and PD1, PDl-1, and BTLA expression; and (iv) reducing VLA-4 expression in both the T- and B-cell populations. Moreover, adoptive transfer of 6-ECDCA– or CDCA-treated donor cells failed to transfer disease in naive recipients. Thus, we show that FXR functions as a negative regulator in neuroinflammation and we highlight that FXR agonists represent a potential previously unidentified therapy for MS. PMID:26811456

  9. Farnesoid X receptor, the bile acid sensing nuclear receptor, in liver regeneration

    PubMed Central

    Li, Guodong; L. Guo, Grace

    2015-01-01

    The liver is unique in regenerative potential, which could recover the lost mass and function after injury from ischemia and resection. The underlying molecular mechanisms of liver regeneration have been extensively studied in the past using the partial hepatectomy (PH) model in rodents, where 2/3 PH is carried out by removing two lobes. The whole process of liver regeneration is complicated, orchestrated event involving a network of connected interactions, which still remain fully elusive. Bile acids (BAs) are ligands of farnesoid X receptor (FXR), a nuclear receptor of ligand-activated transcription factor. FXR has been shown to be highly involved in liver regeneration. BAs and FXR not only interact with each other but also regulate various downstream targets independently during liver regeneration. Moreover, recent findings suggest that tissue-specific FXR also contributes to liver regeneration significantly. These novel findings suggest that FXR has much broader role than regulating BA, cholesterol, lipid and glucose metabolism. Therefore, these researches highlight FXR as an important pharmaceutical target for potential use of FXR ligands to regulate liver regeneration in clinic. This review focuses on the roles of BAs and FXR in liver regeneration and the current underlying molecular mechanisms which contribute to liver regeneration. PMID:26579433

  10. Lysophosphatidic Acid (LPA) Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response1

    PubMed Central

    Shotts, Kristin; Donovan, Erin E.; Strauch, Pamela; Pujanauski, Lindsey M.; Victorino, Francisco; Al-Shami, Amin; Fujiwara, Yuko; Tigyi, Gabor; Oravecz, Tamas; Pelanda, Roberta; Torres, Raul M.

    2014-01-01

    Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. PMID:24890721

  11. Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake.

    PubMed

    Izu, Hanae; Shigemori, Kensuke; Eguchi, Masaya; Kawane, Shuhei; Fujii, Shouko; Kitamura, Yuji; Aoshima, Hitoshi; Yamada, Yasue

    2017-01-01

    We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABAA receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABAA receptor agonist activities. Especially, l-lactic acid showed high agonist activity and its EC50 value was 37μM. Intraperitoneal injections of l-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABAA receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice. PMID:27507485

  12. Identification of Hydroxybenzoic Acids as Selective Lactate Receptor (GPR81) Agonists with Antilipolytic Effects.

    PubMed

    Dvorak, Curt A; Liu, Changlu; Shelton, Jonathan; Kuei, Chester; Sutton, Steven W; Lovenberg, Timothy W; Carruthers, Nicholas I

    2012-08-01

    Following the characterization of the lactate receptor (GPR81), a focused screening effort afforded 3-hydroxybenzoic acid 1 as a weak agonist of both GPR81 and GPR109a (niacin receptor). An examination of structurally similar arylhydroxy acids led to the identification of 3-chloro-5-hydroxybenzoic acid 2, a selective GPR81 agonist that exhibited favorable in vivo effects on lipolysis in a mouse model of obesity.

  13. Identification of Hydroxybenzoic Acids as Selective Lactate Receptor (GPR81) Agonists with Antilipolytic Effects

    PubMed Central

    2012-01-01

    Following the characterization of the lactate receptor (GPR81), a focused screening effort afforded 3-hydroxybenzoic acid 1 as a weak agonist of both GPR81 and GPR109a (niacin receptor). An examination of structurally similar arylhydroxy acids led to the identification of 3-chloro-5-hydroxybenzoic acid 2, a selective GPR81 agonist that exhibited favorable in vivo effects on lipolysis in a mouse model of obesity. PMID:24900524

  14. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120.

    PubMed

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D; Butcher, Adrian J; Ulven, Trond; Miller, Ashley M; Tobin, Andrew B; Milligan, Graeme

    2016-05-01

    It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.

  15. Dominant negative retinoic acid receptor initiates tumor formation in mice

    PubMed Central

    Kupumbati, Tara S; Cattoretti, Giorgio; Marzan, Christine; Farias, Eduardo F; Taneja, Reshma; Mira-y-Lopez, Rafael

    2006-01-01

    Background Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise anti-tumorigenic. Results To address this question, we generated transgenic mice in which expression of a ligand binding defective dominant negative RARα (RARαG303E) was under the control of the mouse mammary tumor virus (MMTV) promoter. The transgene was expressed in the lymphoid compartment and in the mammary epithelium. Observation of aging mice revealed that transgenic mice, unlike their wild type littermates, developed B cell lymphomas at high penetrance, with a median latency of 40 weeks. MMTV-RARαG303E lymphomas were high grade Pax-5+, surface H+L Ig negative, CD69+ and BCL6- and cytologically and phenotypically resembled human adult high grade (Burkitt's or lymphoblastic) lymphomas. We postulated that mammary tumors might arise after a long latency period as seen in other transgenic models of breast cancer. We tested this idea by transplanting transgenic epithelium into the cleared fat pads of wild type hosts, thus bypassing lymphomagenesis. At 17 months post-transplantation, a metastatic mammary adenocarcinoma developed in one of four transplanted glands whereas no tumors developed in sixteen of sixteen endogenous glands with wild type epithelium. Conclusion These findings suggest that physiological RAR activity may normally suppress B lymphocyte and mammary epithelial cell growth and that global RAR inactivation is sufficient to initiate a stochastic process of tumor development requiring multiple transforming events. Our work makes available to the research community a new animal resource that should prove useful as an experimental model of aggressive sporadic lymphoma in immunologically uncompromised hosts. We anticipate that it may also prove useful as a model of breast cancer. PMID

  16. Expression of retinoic acid receptors in human endometrial carcinoma.

    PubMed

    Tanabe, Kojiro; Utsunomiya, Hiroki; Tamura, Mitsutoshi; Niikura, Hitoshi; Takano, Tadao; Yoshinaga, Kohsuke; Nagase, Satoru; Suzuki, Takashi; Ito, Kiyoshi; Matsumoto, Mitsuyo; Hayashi, Shin-ichi; Yaegashi, Nobuo

    2008-02-01

    The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) beta is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RAR beta could act as a tumor suppressor. However, the status of RAR beta in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RAR alpha, RAR beta, and RAR gamma using AM580 (a RAR-specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RAR beta and RAR gamma mRNA was significantly induced by AM580, compared with vehicle controls, whereas RAR alpha mRNA was significantly attenuated by AM580, compared with vehicle. RAR beta was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RAR beta mRNA expression in the Ishikawa cell line, and the expression level of RAR beta in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma.

  17. Structural basis and functions of abscisic acid receptors PYLs

    PubMed Central

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  18. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  19. Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

    PubMed

    Frauli, Mélanie; Neuville, Pascal; Vol, Claire; Pin, Jean-Philippe; Prézeau, Laurent

    2006-02-01

    Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries. PMID:16310227

  20. Omega-3 fatty acids and other FFA4 agonists inhibit growth factor signaling in human prostate cancer cells.

    PubMed

    Liu, Ze; Hopkins, Mandi M; Zhang, Zhihong; Quisenberry, Chrystal B; Fix, Louise C; Galvan, Brianna M; Meier, Kathryn E

    2015-02-01

    Omega-3 fatty acids (n-3 FAs) are proposed to have many beneficial effects on human health. However, the mechanisms underlying their potential cancer preventative effects are unclear. G protein-coupled receptors (GPCRs) of the free fatty acid receptor (FFAR) family, FFA1/GPR40 and FFA4/GPR120, specifically bind n-3 FAs as agonist ligands. In this study, we examined the effects of n-3 FAs in human prostate cancer cell lines. Initial studies established that the long-chain n-3 FAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid, inhibit proliferation of DU145 cells in response to lysophosphatidic acid (LPA), a mitogenic lipid mediator. When added alone to serum-starved DU145 cells, EPA transiently activates signaling events, including p70S6K phosphorylation. However, when added 15 minutes prior to LPA, EPA suppresses LPA-induced activating phosphorylations of ERK, FAK, and p70S6K, and expression of the matricellular protein CCN1. The rapid onset of the inhibitory action of EPA suggested involvement of a GPCR. Further studies showed that DU145 and PC-3 cells express mRNA and protein for both FFA4 and FFA1. TUG-891 (4-[(4-fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid), a selective agonist for FFA4, exerts inhibitory effects on LPA- and epidermal growth factor-induced proliferation and migration, similar to EPA, in DU145 and PC-3 cells. The effects of TUG-891 and EPA are readily reversible. The FFA1/FFA4 agonist GW9508 (4-[[(3-phenoxyphenyl)methyl]amino]-benzenepropranoic acid) likewise inhibits proliferation at doses that block FFA4. Knockdown of FFA4 expression prevents EPA- and TUG-891-induced inhibition of growth and migration. Together, these results indicate that activation of FFA4 initiates signaling events that can inhibit growth factor-induced signaling, providing a novel mechanism for suppression of cancer cell proliferation.

  1. Interaction between retinoid acid receptor-related orphan receptor alpha (RORA) and neuropeptide S receptor 1 (NPSR1) in asthma.

    PubMed

    Acevedo, Nathalie; Sääf, Annika; Söderhäll, Cilla; Melén, Erik; Mandelin, Jami; Pietras, Christina Orsmark; Ezer, Sini; Karisola, Piia; Vendelin, Johanna; Gennäs, Gustav Boije af; Yli-Kauhaluoma, Jari; Alenius, Harri; von Mutius, Erika; Doekes, Gert; Braun-Fahrländer, Charlotte; Riedler, Josef; van Hage, Marianne; D'Amato, Mauro; Scheynius, Annika; Pershagen, Göran; Kere, Juha; Pulkkinen, Ville

    2013-01-01

    Retinoid acid receptor-related Orphan Receptor Alpha (RORA) was recently identified as a susceptibility gene for asthma in a genome-wide association study. To investigate the impact of RORA on asthma susceptibility, we performed a genetic association study between RORA single nucleotide polymorphisms (SNPs) in the vicinity of the asthma-associated SNP (rs11071559) and asthma-related traits. Because the regulatory region of a previously implicated asthma susceptibility gene, Neuropeptide S receptor 1 (NPSR1), has predicted elements for RORA binding, we hypothesized that RORA may interact biologically and genetically with NPSR1. 37 RORA SNPs and eight NPSR1 SNPs were genotyped in the Swedish birth cohort BAMSE (2033 children) and the European cross-sectional PARSIFAL study (1120 children). Seven RORA SNPs confined into a 49 kb region were significantly associated with physician-diagnosed childhood asthma. The most significant association with rs7164773 (T/C) was driven by the CC genotype in asthma cases (OR = 2.0, 95%CI 1.36-2.93, p = 0.0003 in BAMSE; and 1.61, 1.18-2.19, p = 0.002 in the combined BAMSE-PARSIFAL datasets, respectively), and strikingly, the risk effect was dependent on the Gln344Arg mutation in NPSR1. In cell models, stimulation of NPSR1 activated a pathway including RORA and other circadian clock genes. Over-expression of RORA decreased NPSR1 promoter activity further suggesting a regulatory loop between these genes. In addition, Rora mRNA expression was lower in the lung tissue of Npsr1 deficient mice compared to wildtype littermates during the early hours of the light period. We conclude that RORA SNPs are associated with childhood asthma and show epistasis with NPSR1, and the interaction between RORA and NPSR1 may be of biological relevance. Combinations of common susceptibility alleles and less common functional polymorphisms may modify the joint risk effects on asthma susceptibility. PMID:23565190

  2. Therapeutic role of bile acids and nuclear receptor agonists in fibrosing cholangiopathies.

    PubMed

    Trauner, Michael; Halilbasic, Emina; Kazemi-Shirazi, Lili; Kienbacher, Christian; Staufer, Katharina; Traussnigg, Stefan; Hofer, Harald

    2014-01-01

    Chronic inflammatory bile duct diseases such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) result in progressive fibrosis of the biliary tract and ultimately cirrhosis of the liver. Since the etiology and pathogenesis of these fibrosing cholangiopathies are still poorly understood, therapeutic options are rather limited at present. Ursodeoxycholic acid (UDCA) is the paradigm therapeutic bile acid and established standard treatment for PBC, but its role for medical therapy of PSC is still under debate. Promising novel bile acid-based therapeutic options include 24-norursodeoxycholic acid, a side chain-shortened C23 homologue of UDCA, and bile acid receptor/farnesoid X receptor agonists (e.g., obeticholic acid) which currently undergo clinical development for fibrosing cholangiopathies such as PBC and PSC. Other nuclear receptors such as vitamin D receptor and fatty acid-activated peroxisome proliferator-activated receptors are also of considerable interest. This review article is a summary of an overview talk given at Falk Symposium 191 on Advances in Pathogenesis and Treatment of Liver Diseases held in London, October 3-4, 2013, and summarizes the recent progress with novel therapeutic bile acids and bile acid derivatives as novel therapies for fibrosing cholangiopathies such as PBC and PSC.

  3. Effects of beer and hop on ionotropic gamma-aminobutyric acid receptors.

    PubMed

    Aoshima, Hitoshi; Takeda, Katsuichi; Okita, Yoichi; Hossain, Sheikh Julfikar; Koda, Hirofumi; Kiso, Yoshinobu

    2006-04-01

    Beer induced the response of the ionotropic gamma-aminobutyric acid receptors (GABA(A) receptors) expressed in Xenopus oocytes, indicating the presence of gamma-aminobutyric acid (GABA)-like activity. Furthermore, the pentane extract of the beer, hop (Humulus lupulus L.) oil, and myrcenol potentiated the GABA(A) receptor response elicited by GABA. The GABA(A) receptor responses were also potentiated by the addition of aliphatic esters, most of which are reported to be present in beer flavor. Aliphatic esters showed the tendency to decrease in the potentiation of the GABA(A) receptor response with an increase in their carbon chain length. When myrcenol was injected to mice prior to intraperitoneal administration of pentobarbital, the pentobarbital-induced sleeping time of mice increased additionally. Therefore, the beer contained not only GABA-like activity but also the modulator(s) of the GABA(A) receptor response.

  4. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors.

    PubMed

    Takahashi, Akiyoshi; Davis, Perry; Reinick, Christina; Mizusawa, Kanta; Sakamoto, Tatsuya; Dores, Robert M

    2016-06-01

    This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in "Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish" (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR. PMID:27408924

  5. Potentiation of Gamma Aminobutyric Acid Receptors (GABAAR) by Ethanol: How Are Inhibitory Receptors Affected?

    PubMed Central

    Förstera, Benjamin; Castro, Patricio A.; Moraga-Cid, Gustavo; Aguayo, Luis G.

    2016-01-01

    In recent years there has been an increase in the understanding of ethanol actions on the type A γ-aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools. PMID:27199667

  6. Aromatic L-amino acids activate the calcium-sensing receptor.

    PubMed

    Conigrave, Arthur D; Mun, Hee-Chang; Lok, Hiu-Chuen

    2007-06-01

    The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.

  7. Nuclear retinoic acid receptors: conductors of the retinoic acid symphony during development.

    PubMed

    Samarut, Eric; Rochette-Egly, Cécile

    2012-01-30

    The vitamin A derivative, retinoic acid (RA), is essential for embryonic development through the activation of cognate nuclear receptors, RARs, which work as ligand dependent regulators of transcription. In vitro studies revealed how RARs control gene expression at the molecular level and now it appears that it is fine-tuned by a phosphorylation code. In addition, several genetic approaches provided valuable insights on the functions of RARs during development and on the influence of other actors such as the enzymes involved in RA synthesis and degradation and other signaling pathways. It appears that RARs are the conductors of the RA signaling symphony through controlling the dynamics and the coordination of the different players and development steps.

  8. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

    PubMed

    Tunaru, Sorin; Althoff, Till F; Nüsing, Rolf M; Diener, Martin; Offermanns, Stefan

    2012-06-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

  9. γ-Hydroxybutyric acid (GHB) is not an agonist of extrasynaptic GABAA receptors.

    PubMed

    Connelly, William M; Errington, Adam C; Crunelli, Vincenzo

    2013-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  10. Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta.

    PubMed

    Lager, S; Ramirez, V I; Gaccioli, F; Jansson, T; Powell, T L

    2014-07-01

    Fatty acids can function as signaling molecules, acting through receptors in the cytosol or on the cell surface. G-Protein Receptor (GPR)120 is a membrane-bound receptor mediating anti-inflammatory and insulin-sensitizing effects of the omega-3 fatty acid docohexaenoic acid (DHA). GPR120 dysfunction is associated with obesity in humans. Cellular localization of GPR120 and the influence of maternal obesity on GPR120 protein expression in the placenta are unknown. Herein we demonstrate that GPR120 is predominantly expressed in the microvillous membrane (MVM) of human placenta and that the expression level of this receptor in MVM is not altered by maternal body mass index (BMI).

  11. The effects of avermectin on amino acid neurotransmitters and their receptors in the pigeon brain.

    PubMed

    Chen, Li-Jie; Sun, Bao-Hong; Cao, Ye; Yao, Hai-Dong; Qu, Jian-Ping; Liu, Ci; Xu, Shi-Wen; Li, Shu

    2014-03-01

    The objective of this study was to examine the effects of avermectin (AVM) on amino acid neurotransmitters and their receptors in the pigeon brain. Four groups two-month-old American king pigeons (n=20/group) were fed either a commercial diet or an AVM-supplemented diet (20mg/kg·diet, 40 mg/kg·diet, or 60 mg/kg·diet) for 30, 60, or 90 days. The contents of aspartic acid (ASP), glutamate (GLU), glycine (GLY), and γ-aminobutyric acid (GABA) in the brain tissues were determined using ultraviolet high-performance liquid chromatography (HPLC). The expression levels of the GLU and GABA receptor genes were analyzed using real-time quantitative polymerase chain reaction (qPCR). The results indicate that AVM exposure significantly enhances the contents of GABA, GLY, GLU, and ASP in the cerebrum, cerebellum, and optic lobe. In addition, AVM exposure increases the mRNA expression levels of γ-aminobutyric acid type A receptor (GABAAR), γ-aminobutyric acid type B receptor (GABABR), N-methyl-d-aspartate 1 receptor (NR1), N-methyl-d-aspartate 2A receptor (NR2A), and N-methyl-d-aspartate 2B receptor (NR2B) in a dose- and time-dependent manner. Moreover, we found that the most damaged organ was the cerebrum, followed by the cerebellum, and then the optic lobe. These results show that the AVM-induced neurotoxicity may be associated with its effects on amino acid neurotransmitters and their receptors. The information presented in this study will help supplement the available data for future AVM toxicity studies.

  12. Developmental toxicity of perfluorononanoic acid is dependent on peroxisome proliferator activated receptor-alpha.

    EPA Science Inventory

    Perfluorononanoic acid (PFNA) is one of the predominant perfluoroalkyl acids in the environment and in tissues of humans and wildlife. PFNA strongly activates the mouse and human peroxisome proliferator-activated receptor-alpha (PPARα) in vitro and negatively impacts development ...

  13. REACTIVITY PROFILE OF LIGANDS OF MAMMALIAN RETINOIC ACID RECEPTORS: A PRELIMINARY COREPA ANALYSIS

    EPA Science Inventory

    Retinoic acid and associated derivatives comprise a class of endogenous hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of vertebrate development. Identification of potential RAR and RXR ligands is of interes...

  14. Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

    PubMed Central

    1988-01-01

    Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion- promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+- binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins. PMID:2454931

  15. The inimitable kynurenic acid: the roles of different ionotropic receptors in the action of kynurenic acid at a spinal level.

    PubMed

    Tuboly, Gabor; Tar, Lilla; Bohar, Zsuzsanna; Safrany-Fark, Arpad; Petrovszki, Zita; Kekesi, Gabriella; Vecsei, Laszlo; Pardutz, Arpad; Horvath, Gyongyi

    2015-03-01

    Kynurenic acid (KYNA) is a neuroactive metabolite that interacts with NMDA, AMPA/kainate and alpha 7 nicotinic receptors. The goal of this study was to clarify the roles of these receptors in the action of KYNA at a spinal level by using highly specific receptor antagonists alone or in triple combinations. Chronic osteoarthritis-like joint pain was induced with monosodium-iodoacetate in male Wistar rats. Mechanical allodynia and motor function were quantified. In the first series we determined the dose-response and time course effects of intrathecally administered KYNA (10-100 μg), D-(-)-2-amino-5-phosphonopentanoic acid (AP5; an NMDA receptor antagonist; 10-200 μg), methyllycaconitine (MLA; an alpha 7 nicotinic receptor antagonist; 100-200 μg) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX; an AMPA/kainate receptor antagonist; 1-20 μg). In the second series, four different triple combinations of MLA, AP5 and NBQX were investigated. Intrathecal administration of KYNA caused a dose-dependent motor impairment and antinociception. The highly specific NMDA receptor antagonist AP5 caused a motor impairment and antinociception with lower potency. High doses of NBQX resulted in significant antinociception with a slight motor impairment, while only the highest dose of MLA gave rise to significant antinociception with a slight motor impairment. After the coadministration of these ligands as combinations, no potentiation was observed. It may be supposed that the effects of KYNA are primarily due to the inhibition of NMDA receptors at both glycine and phencyclidine (PCP) binding sites, and not to the interactions at the different ionotropic receptors, but the mechanisms behind its high bio-efficiency are still unknown.

  16. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

    SciTech Connect

    Pfeiffer, A.; Rochlitz, H.; Herz, A.; Paumgartner, G. )

    1988-04-01

    The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine with a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.

  17. Stacking interaction and its role in kynurenic acid binding to glutamate ionotropic receptors.

    PubMed

    Zhuravlev, Alexander V; Zakharov, Gennady A; Shchegolev, Boris F; Savvateeva-Popova, Elena V

    2012-05-01

    Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation. PMID:21833825

  18. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid.

    PubMed

    Hwang, Daniel H; Kim, Jeong-A; Lee, Joo Young

    2016-08-15

    Saturated fatty acids can activate Toll-like receptor 2 (TLR2) and TLR4 but polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) inhibit the activation. Lipopolysaccharides (LPS) and lipopetides, ligands for TLR4 and TLR2, respectively, are acylated by saturated fatty acids. Removal of these fatty acids results in loss of their ligand activity suggesting that the saturated fatty acyl moieties are required for the receptor activation. X-ray crystallographic studies revealed that these saturated fatty acyl groups of the ligands directly occupy hydrophobic lipid binding domains of the receptors (or co-receptor) and induce the dimerization which is prerequisite for the receptor activation. Saturated fatty acids also induce the dimerization and translocation of TLR4 and TLR2 into lipid rafts in plasma membrane and this process is inhibited by DHA. Whether saturated fatty acids induce the dimerization of the receptors by interacting with these lipid binding domains is not known. Many experimental results suggest that saturated fatty acids promote the formation of lipid rafts and recruitment of TLRs into lipid rafts leading to ligand independent dimerization of the receptors. Such a mode of ligand independent receptor activation defies the conventional concept of ligand induced receptor activation; however, this may enable diverse non-microbial molecules with endogenous and dietary origins to modulate TLR-mediated immune responses. Emerging experimental evidence reveals that TLRs play a key role in bridging diet-induced endocrine and metabolic changes to immune responses.

  19. Upregulation of retinoic acid receptor-beta by the epidermal growth factor-receptor inhibitor PD153035 is not mediated by blockade of ErbB pathways.

    PubMed

    Grunt, Thomas W; Tomek, Katharina; Wagner, Renate; Puckmair, Klaudia; Kainz, Birgit; Rünzler, Dominik; Gaiger, Alexander; Köhler, Gottfried; Zielinski, Christoph C

    2007-06-01

    Inhibiting epidermal growth factor-receptor (ErbB-1) represents a powerful anticancer strategy. Activation of retinoid pathways is also in development for cancer treatment. Retinoic acid receptor-beta-the tumor suppressor and main retinoid mediator--is silenced in many tumors. The ErbB-1 inhibitor PD153035 cooperates with retinoic acid during growth inhibition and induces retinoic acid receptor-beta suggesting that ErbB-1 controls retinoic acid receptor-beta. However, here we demonstrate that ErbB pathways are not involved in PD153035-mediated retinoic acid receptor-beta-upregulation. PD153035 inhibits ErbB-1-phosphorylation, whereas its derivative EBE-A22 is inactive. Yet both inhibit cell growth and upregulate retinoic acid receptor-beta in ErbB-1-overexpressing (MDA-MB-468), moderately expressing (OVCAR-3), ErbB-1-negative (MDA-MB-453) or ErbB-negative cells (CEM, Jurkat). Both bind DNA, whereas the closely related ErbB-1 inhibitors AG1478 and ZD1839, which are inactive on retinoic acid receptor-beta, do not significantly bind DNA. None of the other ErbB-1/ErbB-2 inhibitors tested (RG-14620, LFM-A12, AG879, AG825) affect retinoic acid receptor-beta. PD153035 decreases methylation of the retinoic acid receptor-beta2 promoter. In OVCAR-3, it stimulates dislodgement of histone deacetylase 1 from the promoter and acetylation of histones H3 and H4. Consequently, PD153035 facilitates recruitment of RNA polymerase II to the promoter and stimulates transcriptional activity. Moreover, PD153035 increases the retinoic acid receptor-beta mRNA half-life. No other retinoid receptor, nor estrogen receptor-alpha, nor RASSF1A is upregulated by PD153035. Thus PD153035 induces retinoic acid receptor-beta by ErbB-independent transcriptional and post-transcriptional mechanisms. This report highlights a triple action for an ErbB-1 inhibitor (ErbB-1 inhibition, DNA intercalation, retinoic acid receptor-beta-induction). Such multitargeting drugs bear great potential for cancer

  20. Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry.

    PubMed

    Li, Xiaoxin; Bangari, Dinesh S; Sharma, Anurag; Mittal, Suresh K

    2009-09-30

    Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin-Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized alpha(2,3)-linked and alpha(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.

  1. [Discovery of potential nicotinic acid receptor agonists from Chinese herbal medicines based on molecular simulation].

    PubMed

    Jiang, Lu-Di; He, Yu-Su; Zhang, Yan-Ling

    2014-12-01

    Nicotinic acid could increase high density lipoprotein and reduce serum total cholesterol, low density lipoprotein cholesterol and triglycerides in human bodies, thus is frequently applied in treating low high-density lipoprotein cholesterol and hypertriglyceridemia in clinic. However, according to the findings, nicotinic acid could also cause adverse effects, such as skin flush, beside its curative effects. In this study, bioisosterism, fragment-based search and Lipinski's Rule of Five were used to preliminarily screen out potential TCM ingredients that may have similar pharmacological effects with nicotinic acid from Traditional Chinese medicine database (TCMD). Afterwards, homology modeling and flexible docking were used to further screen out potential nicotinic acid receptor agonists. As a result, eleven candidate compounds were derived from eight commonly used traditional Chinese medicines. Specifically, all of the candidate compounds' interaction with nicotinic acid receptor was similar to nicotinic acid, and their docking scores were all higher than that of nicotinic acid, but their druggability remained to be further studied. Some of the eight source traditional Chinese medicines were used to lower lipid according to literature studies, implying that they may show effect through above means. In summary, this study provides basis and reference for extracting new nicotinic acid receptor agonists from traditional Chinese medicines and improving the medication status of hyperlipidemia.

  2. A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Lima, Daniela; Pierzchalski, Keely; Jones, Jace W.; Kane, Maureen; Nishikawa, Jun-Ichi; Hiromori, Youhei; Nakanishi, Tsuyoshi; Santos, Miguel M.; Castro, L. Filipe C.; Bourguet, William

    2014-01-01

    Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms. PMID:25116705

  3. Farnesoid X Receptor Agonists and Other Bile Acid Signaling Strategies for Treatment of Liver Disease.

    PubMed

    Halilbasic, Emina; Fuchs, Claudia; Traussnigg, Stefan; Trauner, Michael

    2016-01-01

    The intracellular nuclear receptor farnesoid X receptor (FXR) and the transmembrane G protein-coupled receptor 5 (TGR5) respond to bile acids (BAs) by activating transcriptional networks and/or signaling cascades. These cascades affect the expression of a great number of target genes relevant for BA, cholesterol, lipid and carbohydrate metabolism, as well as genes involved in inflammation, fibrosis and carcinogenesis. FXR activation in the liver tissue and beyond, such as the gut-liver axis, kidney and adipose tissue, plays a role in metabolic diseases. These BA receptors activators hold promise to become a new class of drugs to be used in the treatment of chronic liver disease, hepatocellular cancer and extrahepatic inflammatory and metabolic diseases. This review discusses the relevant BA receptors, the new drugs that target BA transport and signaling and their possible applications. PMID:27332721

  4. Imaging Cancer Cells Expressing the Folate Receptor with Carbon Dots Produced from Folic Acid.

    PubMed

    Bhunia, Susanta Kumar; Maity, Amit Ranjan; Nandi, Sukhendu; Stepensky, David; Jelinek, Raz

    2016-04-01

    Development of new imaging tools for cancer cells in vitro and in vitro is important for advancing cancer research, elucidating drug effects upon cancer cells, and studying cellular processes. We showed that fluorescent carbon dots (C-dots) synthesized from folic acid can serve as an effective vehicle for imaging cancer cells expressing the folate receptor on their surface. The C-dots, synthesized through a simple one-step process from folic acid as the carbon source, exhibited selectivity towards cancer cells displaying the folate receptor, making such cells easily distinguishable in fluorescence microscopy imaging. Biophysical measurements and competition experiments both confirmed the specific targeting and enhanced uptake of C-dots by the folate receptor-expressing cells. The folic acid-derived C-dots were not cytotoxic, and their use in bioimaging applications could aid biological studies of cancer cells, identification of agonists/antagonists, and cancer diagnostics.

  5. TRIP6 Enhances Lysophosphatidic Acid-induced Cell Migration by Interacting with the Lysophosphatidic Acid 2 Receptor*

    PubMed Central

    Xu, Jun; Lai, Yun-Ju; Lin, Weei-Chin; Lin, Fang-Tsyr

    2014-01-01

    Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA2 receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA2 receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130cas in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA2 receptor but not LPA1 or LPA3 receptor, indicating a specific role for TRIP6 in regulating LPA2 receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA2 receptor and downstream signals involved in cell adhesion and migration. PMID:14688263

  6. Glutamate receptor-like channels in plants: a role as amino acid sensors in plant defence?

    PubMed Central

    Roberts, Michael R.

    2014-01-01

    Plant glutamate receptor-like genes (GLRs) are homologous to the genes for mammalian ionotropic glutamate receptors (iGluRs), after which they were named, but in the 16 years since their existence was first revealed, progress in elucidating their biological role has been disappointingly slow. Recently, however, studies from a number of laboratories focusing on the model plant species Arabidopsis thaliana (L.) have thrown new light on the functional properties of some members of the GLR gene family. One important finding has been that plant GLR receptors have a much broader ligand specificity than their mammalian iGluR counterparts, with evidence that some individual GLR receptors can be gated by as many as seven amino acids. These results, together with the ubiquity of their expression throughout the plant, open up the possibility that GLR receptors could have a pervasive role in plants as non-specific amino acid sensors in diverse biological processes. Addressing what one of these roles could be, recent studies examining the wound response and disease susceptibility in GLR knockout mutants have provided evidence that some members of clade 3 of the GLR gene family encode important components of the plant's defence response. Ways in which this family of amino acid receptors might contribute to the plant's ability to respond to an attack from pests and pathogens are discussed. PMID:24991414

  7. The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation.

    PubMed

    Pols, Thijs W H; Noriega, Lilia G; Nomura, Mitsunori; Auwerx, Johan; Schoonjans, Kristina

    2011-06-01

    Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids, and their levels fluctuate in the intestine as well as in the blood circulation depending on food intake. Besides their role in dietary lipid absorption, bile acids function as signaling molecules capable to activate specific receptors. These BA receptors are not only important in the regulation of bile acid synthesis and their metabolism, but also regulate glucose homeostasis, lipid metabolism, and energy expenditure. These processes are important in diabetes and other facets of the metabolic syndrome, which represents a considerable increasing health burden. In addition to the function of the nuclear receptor FXRα in regulating local effects in the organs of the enterohepatic axis, increasing evidence points to a crucial role of the G-protein coupled receptor (GPCR) TGR5 in mediating systemic actions of BAs. Here we discuss the current knowledge on BA receptors, with a strong focus on the cell membrane receptor TGR5, which emerges as a valuable target for intervention in metabolic diseases. PMID:21145931

  8. Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation.

    PubMed

    Sánchez-Reyes, Omar B; Romero-Ávila, M Teresa; Castillo-Badillo, Jean A; Takei, Yoshinori; Hirasawa, Akira; Tsujimoto, Gozoh; Villalobos-Molina, Rafael; García-Sáinz, J Adolfo

    2014-01-15

    GPR120, free fatty acid receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in intracellular calcium) and phosphorylation were studied. It was observed that the fusion protein migrated in sodium dodecyl sulfate-polyacrylamide gels as a band with a mass of ≈70-75kDa, although other bands of higher apparent weight (>130kDa) were also detected. Cell stimulation with docosahexaenoic acid or α-linolenic acid induced concentration-dependent increases in intracellular calcium and GPR120 phosphorylation. Activation of protein kinase C with phorbol esters also induced a marked receptor phosphorylation but did not alter the ability of 1µM docosahexaenoic acid to increase the intracellular calcium concentration. Phorbol ester-induced GPR120 phosphorylation, but not that induced with docosahexaenoic acid, was blocked by protein kinase C inhibitors (bis-indolyl-maleimide I and Gö 6976) suggesting that conventional kinase isoforms mediate this action. The absence of effect of protein kinase C inhibitors on agonist-induced GPR120 phosphorylation indicates that this kinase does not play a major role in agonist-induced receptor phosphorylation. Docosahexaenoic acid action was associated with marked GPR120 internalization whereas that induced with phorbol esters was smaller at early times. PMID:24239485

  9. Statistical Mechanics Model for the Interaction between the Neurotransmitter γ-Aminobutyric acid and GABAA Receptors

    NASA Astrophysics Data System (ADS)

    Zafar, Sufi; Saxena, Nina C.; Conrad, Kevin A.; Hussain, Arif

    2004-07-01

    Interactions between the neurotransmitter γ-aminobutyric acid (GABA) and GABAA receptor ion channels play an important role in the central nervous system. A statistical mechanics model is proposed for the interaction between GABA and GABAA receptors. The model provides good fits to the electrophysiology data as well as an estimation of receptor activation energies, and predicts the temperature dependence consistent with measurements. In addition, the model provides insights into single channel conductance measurements. This model is also applicable to other ligand-gated ion channels with similar pentameric structures.

  10. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.

    PubMed

    Ye, Shixin; Köhrer, Caroline; Huber, Thomas; Kazmi, Manija; Sachdev, Pallavi; Yan, Elsa C Y; Bhagat, Aditi; RajBhandary, Uttam L; Sakmar, Thomas P

    2008-01-18

    G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors. PMID:17993461

  11. A differential fluorescent receptor for nucleic acid analysis.

    PubMed

    Bengtson, Hillary N; Kolpashchikov, Dmitry M

    2014-01-24

    Differential receptors use an array of sensors to recognize analytes. Each sensor in the array can recognize not one, but several analytes with different rates, so a single analyte triggers a response of several sensors in the array. The receptor thus produces a pattern of signals that is unique for each analyte, thereby enabling identification of a specific analyte by producing a "fingerprint" pattern. We applied this approach for the analysis of DNA sequences of Mycobacterium tuberculosis strains that differ by single nucleotide substitutions in the 81-bp hot-spot region that imparts rifampin resistance. The technology takes advantage of the new multicomponent, selfassembling sensor, which produces a fluorescent signal in the presence of specific DNA sequences. A differential fluorescent receptor (DFR) contained an array of three such sensors and differentiated at least eight DNA sequences. The approach requires only one molecular-beacon-like fluorescent reporter, which can be used by all three sensors. The DFR developed in this study represents a cost-efficient alternative to molecular diagnostic technologies that use fluorescent hybridization probes.

  12. Recent Progress on Bile Acid Receptor Modulators for Treatment of Metabolic Diseases.

    PubMed

    Xu, Yanping

    2016-07-28

    Bile acids are steroid-derived molecules synthesized in the liver, secreted from hepatocytes into the bile canaliculi, and subsequently stored in the gall bladder. During the feeding, bile flows into the duodenum, where it contributes to the solubilization and digestion of lipid-soluble nutrients. After a meal, bile-acid levels increase in the intestine, liver, and also in the systemic circulation. Therefore, serum bile-acid levels serve as an important sensing mechanism for nutrient and energy. Recent studies have described bile acids as versatile signaling molecules endowed with systemic endocrine functions. Bile acids are ligands for G-protein coupled receptors (GPCRs) such as TGR5 (also known as GPBAR1, M-BAR, and BG37) and nuclear hormone receptors including farnesoid X receptor (FXR; also known as NR1H4). Acting through these diverse signaling pathways, bile acids regulate triglyceride, cholesterol, glucose homeostasis, and energy expenditure. These bile-acid-controlled signaling pathways have become the source of promising novel drug targets to treat common metabolic and hepatic diseases. PMID:26878262

  13. Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor α (RORα)

    PubMed Central

    Crumbley, Christine; Wang, Yongjun; Banerjee, Subhashis; Burris, Thomas P.

    2012-01-01

    The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression. PMID:22485150

  14. Amiloride inhibition of gamma-aminobutyric acid(A) receptors depends upon the alpha subunit subtype.

    PubMed

    Fisher, Janet L

    2002-06-01

    gamma-Aminobutyric acid(A) (GABA(A)) receptors (GABARs) are responsible for most fast inhibitory neurotransmission in the mammalian brain. The GABARs contain several allosteric modulatory sites, many of which are useful clinically. The activity of most of these modulators depends upon the subunit composition of the receptor. The diuretic amiloride was previously reported to inhibit GABARs in frog sensory neurons. We measured its effects on recombinant GABARs to determine its mechanism of action at mammalian receptors and to examine the effect of subunit composition. Amiloride acted primarily as a competitive antagonist, reducing the sensitivity of the receptor to GABA without affecting the maximal current amplitude. Receptors containing an alpha6 subunit were about 10-fold more sensitive to amiloride than those containing other alpha subunits. In contrast, the identity of the beta or gamma subtype had little effect on amiloride sensitivity. Although several other modulators have specific effects at alpha6-containing receptors, amiloride is the first inhibitor to be reported with no additional dependence on the identity of the beta or gamma subunit. Therefore, it probably represents a unique modulatory site on the GABAR, which could be useful for developing drugs targeting these receptors. The selective activity of amiloride could also be helpful for isolating the contribution of receptors composed of alpha6 subtypes in heterogeneous native GABAR populations.

  15. Structural basis for molecular recognition of folic acid by folate receptors.

    PubMed

    Chen, Chen; Ke, Jiyuan; Zhou, X Edward; Yi, Wei; Brunzelle, Joseph S; Li, Jun; Yong, Eu-Leong; Xu, H Eric; Melcher, Karsten

    2013-08-22

    Folate receptors (FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate cellular uptake of folate. Although expressed at very low levels in most tissues, folate receptors, especially FRα, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions. The folate dependency of many tumours has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, high-affinity antifolates, folate-based imaging agents and folate-conjugated drugs and toxins. To understand how folate binds its receptors, we determined the crystal structure of human FRα in complex with folic acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulphide bonds and contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for designing more specific drugs targeting the folate receptor system.

  16. Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

    PubMed

    Michaille, J J; Blanchet, S; Kanzler, B; Garnier, J M; Dhouailly, D

    1994-12-01

    Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

  17. Minireview: The Effects of Species Ortholog and SNP Variation on Receptors for Free Fatty Acids

    PubMed Central

    Hudson, Brian D.; Murdoch, Hannah

    2013-01-01

    Although it is widely assumed that species orthologs of hormone-responsive G protein-coupled receptors will be activated by the same endogenously produced ligand(s), variation in potency, particularly in cases in which more than 1 receptor responds to the same hormone, can result in challenges in defining the contribution of individual receptors in different species. This can create considerably greater issues when using synthetic chemical ligands and, in some cases, may result in a complete lack of efficacy of such a ligand when used in animal models of pathophysiology. In man, the concept that distinct responses of individuals to medicines may reflect differences in the ability of such drugs to bind to or activate single nucleotide polymorphism variants of receptors is more established as a concept but, in many cases, clear links between such variants that are associated with disease phenotypes and substantial differences in receptor ligand pharmacology have been more difficult to obtain. Herein we consider each of these issues for the group of free fatty acid receptors, FFA1-FFA4, defined to be activated by free fatty acids of varying chain length, which, based on their production by 1 tissue or location and action in distinct locations, have been suggested to possess characteristics of hormones. PMID:23686113

  18. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein–Coupled Bile Acid Receptors

    PubMed Central

    Brighton, Cheryl A.; Rievaj, Juraj; Kuhre, Rune E.; Glass, Leslie L.; Schoonjans, Kristina; Holst, Jens J.

    2015-01-01

    Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein–coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1–secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca2+. In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca2+ response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber–mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms. PMID:26280129

  19. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors.

    PubMed

    Brighton, Cheryl A; Rievaj, Juraj; Kuhre, Rune E; Glass, Leslie L; Schoonjans, Kristina; Holst, Jens J; Gribble, Fiona M; Reimann, Frank

    2015-11-01

    Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.

  20. Cross-talk between lysophosphatidic acid receptor 1 and tropomyosin receptor kinase A promotes lung epithelial cell migration.

    PubMed

    Nan, Ling; Wei, Jianxin; Jacko, Anastasia M; Culley, Miranda K; Zhao, Jing; Natarajan, Viswanathan; Ma, Haichun; Zhao, Yutong

    2016-02-01

    Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which plays a crucial role in the regulation of cell proliferation, migration, and differentiation. LPA exerts its biological effects mainly through binding to cell-surface LPA receptors (LPA1-6), which belong to the G protein-coupled receptor (GPCR) family. Recent studies suggest that cross-talk between receptor tyrosine kinases (RTKs) and GPCRs modulates GPCRs-mediated signaling. Tropomyosin receptor kinase A (TrkA) is a RTK, which mediates nerve growth factor (NGF)-induced biological functions including cell migration in neuronal and non-neuronal cells. Here, we show LPA1 transactivation of TrkA in murine lung epithelial cells (MLE12). LPA induced tyrosine phosphorylation of TrkA in both time- and dose-dependent manners. Down-regulation of LPA1 by siRNA transfection attenuated LPA-induced phosphorylation of TrkA, suggesting a cross-talk between LPA1 and TrkA. To investigate the molecular regulation of the cross-talk, we focused on the interaction between LPA1 and TrkA. We found that LPA induced interaction between LPA1 and TrkA. The LPA1/TrkA complex was localized on the plasma membrane and in the cytoplasm. The C-terminus of LPA1 was identified as the binding site for TrkA. Inhibition of TrkA attenuated LPA-induced phosphorylation of TrkA and LPA1 internalization, as well as lung epithelial cell migration. These studies provide a molecular mechanism for the transactivation of TrkA by LPA, and suggest that the cross-talk between LPA1 and TrkA regulates LPA-induced receptor internalization and lung epithelial cell migration. PMID:26597701

  1. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    PubMed

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  2. Identification of dehydroabietc acid from Boswellia thurifera resin as a positive GABAA receptor modulator.

    PubMed

    Rueda, Diana C; Raith, Melanie; De Mieri, Maria; Schöffmann, Angela; Hering, Steffen; Hamburger, Matthias

    2014-12-01

    In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes, a petroleum ether extract (100 μg/mL) of the resin of Boswellia thurifera (Burseraceae) potentiated GABA-induced chloride currents (IGABA) through receptors of the subtype α₁β₂γ₂s by 319.8% ± 79.8%. With the aid of HPLC-based activity profiling, three known terpenoids, dehydroabietic acid (1), incensole (2), and AKBA (3), were identified in the active fractions of the extract. Structure elucidation was achieved by means of HR-MS and microprobe 1D/2D NMR spectroscopy. Compound 1 induced significant receptor modulation in the oocyte assay, with a maximal potentiation of IGABA of 397.5% ± 34.0%, and EC₅₀ of 8.7 μM ± 1.3 μM. This is the first report of dehydroabietic acid as a positive GABAA receptor modulator. PMID:25200370

  3. Binding of retinoic acid receptor heterodimers to DNA. A role for histones NH2 termini.

    PubMed

    Lefebvre, P; Mouchon, A; Lefebvre, B; Formstecher, P

    1998-05-15

    The retinoic acid signaling pathway is controlled essentially through two types of nuclear receptors, RARs and RXRs. Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor (RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid response element (RARE). Although unliganded RXR/RAR heterodimers bind constitutively to DNA in vitro, a clear in vivo ligand-dependent occupancy of the RARE present in the RARbeta2 gene promoter has been reported (Dey, A., Minucci, S., and Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201). Nucleosomes are viewed as general repressors of the transcriptional machinery, in part by preventing the access of transcription factors to DNA. The ability of hRXRalpha/hRARalpha heterodimers to bind to a nucleosomal template in vitro has therefore been examined. The assembly of a fragment from the RARbeta2 gene promoter, which contains a canonical DR5 RARE, into a nucleosome core prevented hRXRalpha/hRARalpha binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXRalpha/hRARalpha heterodimers. These data establish therefore the role of histones NH2 termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.

  4. Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor-selective retinoic acid analogs.

    PubMed

    Brooks, S C; Kazmer, S; Levin, A A; Yen, A

    1996-01-01

    The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

  5. Γ-aminobutyric acid receptors affect the progression and migration of tumor cells.

    PubMed

    Zhang, Xiaoxue; Du, Zuoyi; Liu, Jun; He, Jianxing

    2014-12-01

    Γ-aminobutyric acid (GABA) is a multifunctional molecule found in the nervous system and non-neuronal tissues. GABA receptors combine with GABA molecules and transmit signal stimuli into cells. In addition to traditional neurotransmission and regulation of secretion, GABA and GABA receptors are involved in cell differentiation and proliferation throughout peripheral organs, as well as in tumorigenesis. The exact mechanism of the GABAergic system in regulating tumor development is unclear, but many studies have revealed that GABA receptors exert critical regulative effects on tumor cell proliferation and migration. In this review, the molecular structure, distribution and biological function of GABA receptors associated with tumorigenesis are described. Recent advances in the elucidation of mechanisms underlying GABAergic signaling control over tumor growth are also discussed.

  6. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

    PubMed Central

    Hopf, Thomas A.; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S.; Benton, Richard

    2015-01-01

    Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection. PMID:25584517

  7. Specificity of the antibody receptor site to D-lysergamide: model of a physiological receptor for lysergic acid diethylamide.

    PubMed

    Van Vunakis, H; Farrow, J T; Gjika, H B; Levine, L

    1971-07-01

    Antibodies to D-lysergic acid have been produced in rabbits and guinea pigs and a radioimmunoassay for the hapten was developed. The specificity of this lysergamide-antilysergamide reaction was determined by competitive binding with unlabeled lysergic acid diethylamide (LSD), psychotomimetic drugs, neurotransmitters, and other compounds with diverse structures. LSD and several related ergot alkaloids were potent competitors, three to seven times more potent than lysergic acid itself. The N,N-dimethyl derivatives of several compounds, including tryptamine, 5-hydroxytryptamine, 4-hydroxytryptamine, 5-methoxytryptamine, tyramine, and mescaline, were only about ten times less effective than lysergic acid, even though these compounds lack some of the ring systems of lysergic acid. The pattern of inhibition by related compounds with various substituents suggests that the antibody receptor site recognizes structural features resembling the LSD molecule. In particular, the aromatic nucleus and the dimethylated ethylamine side chain in phenylethylamine and tryptamine derivatives may assume in solution a conformation resembling ring A and the methylated nitrogen in ring C of LSD. Among the tryptamine derivatives, a large percentage of the most potent competitors are also psychotomimetic compounds.

  8. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4.

    PubMed

    Lager, Susanne; Gaccioli, Francesca; Ramirez, Vanessa I; Jones, Helen N; Jansson, Thomas; Powell, Theresa L

    2013-03-01

    Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸB, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.

  9. Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

    PubMed Central

    Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Campos-Martínez, Gisselle A.; Meizoso-Huesca, Aldo; García-Sáinz, J. Adolfo

    2015-01-01

    Results The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes. Conclusion Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes. PMID:26473723

  10. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    SciTech Connect

    Junker, L.H.; Davis, R.A. )

    1989-12-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of (14C)cholesterol from (2-14C)acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of (14C)cholesterol from (2-14C)acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

  11. Intracrine prostaglandin E(2) signalling regulates hypoxia-inducible factor-1α expression through retinoic acid receptor-β.

    PubMed

    Fernández-Martínez, Ana B; Jiménez, María I Arenas; Manzano, Victoria Moreno; Lucio-Cazaña, Francisco J

    2012-12-01

    We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-β up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-β and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1β increased retinoic acid receptor-β expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-β antagonist LE-135 or siRNA retinoic acid receptor-β and (iii) there was direct binding of retinoic acid receptor-β to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E(2). Since intracellular prostaglandin E(2) mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-β in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1β and 16,16-dimethyl-prostaglandin E(2) by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-β expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-β signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-β expression is found to be strictly regulated by intracellular prostaglandin E(2). Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production

  12. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells. PMID:25881750

  13. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells.

  14. The NHR-8 nuclear receptor regulates cholesterol and bile acid homeostasis in C. elegans.

    PubMed

    Magner, Daniel B; Wollam, Joshua; Shen, Yidong; Hoppe, Caroline; Li, Dongling; Latza, Christian; Rottiers, Veerle; Hutter, Harald; Antebi, Adam

    2013-08-01

    Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.

  15. Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling.

    PubMed Central

    McGary, C T; Raja, R H; Weigel, P H

    1989-01-01

    Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a

  16. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells.

    PubMed

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  17. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

    PubMed Central

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  18. Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis

    PubMed Central

    Gomez-Ospina, Natalia; Potter, Carol J.; Xiao, Rui; Manickam, Kandamurugu; Kim, Mi-Sun; Kim, Kang Ho; Shneider, Benjamin L.; Picarsic, Jennifer L.; Jacobson, Theodora A.; Zhang, Jing; He, Weimin; Liu, Pengfei; Knisely, A. S.; Finegold, Milton J.; Muzny, Donna M.; Boerwinkle, Eric; Lupski, James R.; Plon, Sharon E.; Gibbs, Richard A.; Eng, Christine M.; Yang, Yaping; Washington, Gabriel C.; Porteus, Matthew H.; Berquist, William E.; Kambham, Neeraja; Singh, Ravinder J.; Xia, Fan; Enns, Gregory M.; Moore, David D.

    2016-01-01

    Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection. PMID:26888176

  19. Evidence for impaired retinoic acid receptor-thyroid hormone receptor AF-2 cofactor activity in human lung cancer.

    PubMed Central

    Moghal, N; Neel, B G

    1995-01-01

    Retinoic acid (RA) is required for normal airway epithelial cell growth and differentiation both in vivo and in vitro. One of the earliest events following the exposure of bronchial epithelial cells to RA is the strong induction of RA receptor beta (RAR beta) mRNA. Previous work established that many lung cancer cell lines and primary tumors display abnormal RAR beta mRNA expression, most often absence or weak expression of the RAR beta 2 isoform, even after RA treatment. Restoration of RAR beta 2 into RAR beta-negative lung cancer cell lines has been reported to inhibit tumorigenicity. Since RAR beta 2 inactivation may contribute to lung cancer, we have investigated the molecular mechanism of defective RAR beta 2 expression. Nuclear run-on assays and transient transfections with RAR beta 2 promoter constructs indicate the presence of trans-acting transcriptional defects in most lung cancer cell lines, which map to the RA response element (RARE). These defects cannot be complemented by RAR-retinoid X receptor cotransfection and can be separated into two types: (i) one affecting transcription from direct repeat RAREs, but not palindromic RAREs, and (ii) another affecting transcription from both types of RARE. Studies using chimeras between RAR alpha, TR alpha, and other transcription factors suggest the existence of novel RAR-thyroid hormone receptor AF-2-specific cofactors, which are necessary for high levels of transcription. Furthermore, these factors may be frequently inactivated in human lung cancer. PMID:7791800

  20. Molecular recognition of amino acids with some fluorescent ditopic pyrylium- and pyridinium-based crown ether receptors

    NASA Astrophysics Data System (ADS)

    Moghimi, A.; Maddah, B.; Yari, A.; Shamsipur, M.; Boostani, M.; Fall Rastegar, M.; Ghaderi, A. R.

    2005-10-01

    The molecular recognition of L-amino acids such as asparagine, glutamine, lysine and arginine with some crownpyryliums, CP's, and a crownpyridinium compound, as receptors, were examined in methanol. 1H NMR spectroscopy was used to examine the structural stability of the receptors in the presence of the amino acids. The fluorimetric titration of the receptors by specified amino acids, other than arginine, was followed within a few minutes and the stoichiometry and stability of the resulting amino acid complexes were evaluated. The data analysis clearly demonstrated the critical role of the terminal amino group to carboxylic acid distance of amino acids for their proper fixation on the receptor molecules. Ion pairing for the two oppositely charged carboxylate anion and pyrylium (or pyridinium) cation, as well as the hydrogen bonding between crown ethers' oxygens and ammonium hydrogens are expected as the main interaction sources in the host-guest complexations.

  1. Peripheral tackykinin and excitatory amino acid receptors mediate hyperalgesia induced by Phoneutria nigriventer venom.

    PubMed

    Zanchet, Eliane Maria; Cury, Yara

    2003-04-25

    The generation of hyperalgesia by Phoneutria nigriventer venom was investigated in rats using the paw pressure test, through the intraplantar injection of the venom. Hyperalgesia was significantly inhibited by N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine), a vanilloid receptor antagonist, by the local administration of pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Pro (spiro-gamma-lactam) Leu-Trp-NH(2) (GR82334) or of Phenyl-CO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH(2) (GR94800), inhibitors of tachykinin NK(1) and NK(2) receptors, respectively, or by the local injection of dizocilpine (MK 801), (+/-)-2-amino-5-phosphonopentanoic acid ((+/-)-AP-5), or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and non-NMDA excitatory amino acid receptors. The correlation between hyperalgesia and the inflammatory response induced by the venom was also investigated. The venom-induced edematogenic response was not modified by the pharmacological treatments. These results suggest that hyperalgesia induced by P. nigriventer venom is mediated by stimulation of capsaicin-sensitive neurons, with activation of peripheral tachykinin NK(1) and NK(2) receptors and of both the NMDA and AMPA receptors. Distinct mechanisms are involved in the development of hyperalgesia and edema induced by the venom.

  2. Signaling through retinoic acid receptors in cardiac development: Doing the right things at the right times.

    PubMed

    Xavier-Neto, José; Sousa Costa, Ângela M; Figueira, Ana Carolina M; Caiaffa, Carlo Donato; Amaral, Fabio Neves do; Peres, Lara Maldanis Cerqueira; da Silva, Bárbara Santos Pires; Santos, Luana Nunes; Moise, Alexander R; Castillo, Hozana Andrade

    2015-02-01

    Retinoic acid (RA) is a terpenoid that is synthesized from vitamin A/retinol (ROL) and binds to the nuclear receptors retinoic acid receptor (RAR)/retinoid X receptor (RXR) to control multiple developmental processes in vertebrates. The available clinical and experimental data provide uncontested evidence for the pleiotropic roles of RA signaling in development of multiple embryonic structures and organs such eyes, central nervous system, gonads, lungs and heart. The development of any of these above-mentioned embryonic organ systems can be effectively utilized to showcase the many strategies utilized by RA signaling. However, it is very likely that the strategies employed to transfer RA signals during cardiac development comprise the majority of the relevant and sophisticated ways through which retinoid signals can be conveyed in a complex biological system. Here, we provide the reader with arguments indicating that RA signaling is exquisitely regulated according to specific phases of cardiac development and that RA signaling itself is one of the major regulators of the timing of cardiac morphogenesis and differentiation. We will focus on the role of signaling by RA receptors (RARs) in early phases of heart development. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

  3. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    SciTech Connect

    Shibata, Kenji . E-mail: kshibata@kyowa.co.jp; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki . E-mail: hirayama@is.icc.u-tokai.ac.jp

    2006-03-10

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with {beta}-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay.

  4. Computer-aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

    NASA Astrophysics Data System (ADS)

    Silva, Carlos H. T. P.; Leopoldino, Andreia M.; Silva, Eloiza H. T.; Espinoza, V. A. A.; Taft, C. A.

    The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand-binding domain contains the ligand-dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6-31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor.

  5. Structure-dependent effects of pyridine derivatives on mechanisms of intestinal fatty acid uptake: regulation of nicotinic acid receptor and fatty acid transporter expression.

    PubMed

    Riedel, Annett; Lang, Roman; Rohm, Barbara; Rubach, Malte; Hofmann, Thomas; Somoza, Veronika

    2014-07-01

    Pyridines are widely distributed in foods. Nicotinic acid (NA), a carboxylated pyridine derivative, inhibits lipolysis in adipocytes by activation of the orphan NA receptor (HM74A) and is applied to treat hyperlipidemia. However, knowledge on the impact of pyridine derivatives on intestinal lipid metabolism is scarce. This study was performed to identify the structural determinants of pyridines for their effects on fatty acid uptake in enterocyte-like Caco-2 cells and to elucidate the mechanisms of action. The impact of 17 pyridine derivatives on fatty acid uptake was tested. Multiple regression analysis revealed the presence of a methyl group to be the structural determinant at 0.1 mM, whereas at 1 mM, the presence of a carboxylic group and the N-methylation presented further structural characteristics to affect the fatty acid uptake. NA, showing a stimulating effect on FA uptake, and N-methyl-4-phenylpyridinium (MPP), inhibiting FA uptake, were selected for mechanistic studies. Gene expression of the fatty acid transporters CD36, FATP2 and FATP4, and the lipid metabolism regulating transcription factors peroxisome proliferator-activated receptor (PPAR) α and PPARγ was up-regulated upon NA treatment. Caco-2 cells were demonstrated to express the low-affinity NA receptor HM74 of which the gene expression was up-regulated upon NA treatment. We hypothesize that the NA-induced fatty acid uptake might result from NA receptor activation and related intracellular signaling cascades. In contrast, MPP increased transepithelial electrical resistance. We therefore conclude that NA and MPP, both sharing the pyridine motif core, exhibit their contrary effects on intestinal FA uptake by activation of different mechanisms.

  6. Structure-dependent effects of pyridine derivatives on mechanisms of intestinal fatty acid uptake: regulation of nicotinic acid receptor and fatty acid transporter expression.

    PubMed

    Riedel, Annett; Lang, Roman; Rohm, Barbara; Rubach, Malte; Hofmann, Thomas; Somoza, Veronika

    2014-07-01

    Pyridines are widely distributed in foods. Nicotinic acid (NA), a carboxylated pyridine derivative, inhibits lipolysis in adipocytes by activation of the orphan NA receptor (HM74A) and is applied to treat hyperlipidemia. However, knowledge on the impact of pyridine derivatives on intestinal lipid metabolism is scarce. This study was performed to identify the structural determinants of pyridines for their effects on fatty acid uptake in enterocyte-like Caco-2 cells and to elucidate the mechanisms of action. The impact of 17 pyridine derivatives on fatty acid uptake was tested. Multiple regression analysis revealed the presence of a methyl group to be the structural determinant at 0.1 mM, whereas at 1 mM, the presence of a carboxylic group and the N-methylation presented further structural characteristics to affect the fatty acid uptake. NA, showing a stimulating effect on FA uptake, and N-methyl-4-phenylpyridinium (MPP), inhibiting FA uptake, were selected for mechanistic studies. Gene expression of the fatty acid transporters CD36, FATP2 and FATP4, and the lipid metabolism regulating transcription factors peroxisome proliferator-activated receptor (PPAR) α and PPARγ was up-regulated upon NA treatment. Caco-2 cells were demonstrated to express the low-affinity NA receptor HM74 of which the gene expression was up-regulated upon NA treatment. We hypothesize that the NA-induced fatty acid uptake might result from NA receptor activation and related intracellular signaling cascades. In contrast, MPP increased transepithelial electrical resistance. We therefore conclude that NA and MPP, both sharing the pyridine motif core, exhibit their contrary effects on intestinal FA uptake by activation of different mechanisms. PMID:24767308

  7. PLZF is a negative regulator of retinoic acid receptor transcriptional activity.

    PubMed

    Martin, Perrine J; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2003-09-01

    BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. CONCLUSION: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled.

  8. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

    PubMed Central

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. PMID:26861299

  9. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling.

    PubMed

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)-AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K-AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. PMID:26861299

  10. Lysergic acid diethylamide-induced Fos expression in rat brain: role of serotonin-2A receptors.

    PubMed

    Gresch, P J; Strickland, L V; Sanders-Bush, E

    2002-01-01

    Lysergic acid diethylamide (LSD) produces altered mood and hallucinations in humans and binds with high affinity to serotonin-2A (5-HT(2A)) receptors. Although LSD interacts with other receptors, the activation of 5-HT(2A) receptors is thought to mediate the hallucinogenic properties of LSD. The goal of this study was to identify the brain sites activated by LSD and to determine the influence of 5-HT(2A) receptors in this activation. Rats were pretreated with the 5-HT(2A) receptor antagonist MDL 100907 (0.3 mg/kg, i.p.) or vehicle 30 min prior to LSD (500 microg/kg, i.p.) administration and killed 3 h later. Brain tissue was examined for Fos protein expression by immunohistochemistry. LSD administration produced a five- to eight-fold increase in Fos-like immunoreactivity in medial prefrontal cortex, anterior cingulate cortex, and central nucleus of amygdala. However, in dorsal striatum and nucleus accumbens no increase in Fos-like immunoreactivity was observed. Pretreatment with MDL 100907 completely blocked LSD-induced Fos-like immunoreactivity in medial prefrontal cortex and anterior cingulate cortex, but only partially blocked LSD-induced Fos-like immunoreactivity in amygdala. Double-labeled immunohistochemistry revealed that LSD did not induce Fos-like immunoreactivity in cortical cells expressing 5-HT(2A) receptors, suggesting an indirect activation of cortical neurons. These results indicate that the LSD activation of medial prefrontal cortex and anterior cingulate cortex is mediated by 5-HT(2A) receptors, whereas in amygdala 5-HT(2A) receptor activation is a component of the response. These findings support the hypothesis that the medial prefrontal cortex, anterior cingulate cortex, and perhaps the amygdala, are important regions involved in the production of hallucinations.

  11. Two isoforms of Xenopus retinoic acid receptor gamma 2 (B) exhibit differential expression and sensitivity to retinoic acid during embryogenesis.

    PubMed

    Crawford, M J; Liversage, R A; Varmuza, S L

    1995-01-01

    We report the isolation of two retinoic acid receptor isoforms (RAR gamma), which differ only in the 5'untranslated and putative N-terminus A regions. The two isoforms appear to serve as early markers for the presumptive neural axis; however, their expression patterns differ. RAR-gamma 2.1 is first expressed at gastrulation at the dorsal lip and subsequently along the presumptive neural axis. RAR- gamma 2.2 represents the full-length sequence of a receptor cDNA already partially characterized and present as a maternal transcript [Ellinger-Ziegelbauer and Dreyer (1991); Genes Dev 5:94-104, (1993): Mech Dev 41:31-46; Pfeffer and DeRobertis, (1994) Mech Dev: 45:147-153]. Unlike RAR-gamma 2.2, the 2.1 variant is not expressed either in pre-somitic mesoderm or notochord. RAR-gamma 2.1 is strongly expressed in branchial arches and to a lesser extent in the neural floor plate. The two isoforms also exhibit differential sensitivity to retinoic acid. Constitutive expression of RAR gamma 2.2 following neurulation appears to be depressed by treatment with retinoic acid, but domains of highest expression, namely, the head and tail, remain relatively unaffected, as do patterns of expression prior to late neurulation. By contrast, RAR-gamma 2.1 is not transcribed in retinoid-inhibited structures. Using microinjection techniques, we show that changes of RAR-gamma 2.1 expression in presumptive head structures occur as an early and local consequence of retinoic acid administration. Since RAR-gamma 2.1 expression is inhibited by retinoic acid, we tested to see if other treatments that perturb axis formation had any effect. Surprisingly, UV irradiation did not suppress that its inhibition by retinoic acid is not due solely to inhibition of anterior neural development. These experiments demonstrate a new subdivision of isoforms that undergo differential expression during development and that exhibit differential sensitivity to retinoic acid and to UV. This sensitivity and the presence

  12. Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

    PubMed Central

    van Blitterswijk, W J; Hilkmann, H

    1993-01-01

    Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation. Images PMID:8392931

  13. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism

    PubMed Central

    Stäubert, Claudia; Broom, Oliver Jay; Nordström, Anders

    2015-01-01

    Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them. We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation. We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death. Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy. PMID:25839160

  14. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism.

    PubMed

    Stäubert, Claudia; Broom, Oliver Jay; Nordström, Anders

    2015-08-14

    Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.

  15. Unsurmountable antagonism of brain 5-hydroxytryptamine2 receptors by (+)-lysergic acid diethylamide and bromo-lysergic acid diethylamide.

    PubMed

    Burris, K D; Sanders-Bush, E

    1992-11-01

    Lysergic acid diethylamide (LSD) and its structural analogue 2-bromo-lysergic acid diethylamide (BOL) act as unsurmountable antagonists of serotonin-elicited contractions in smooth muscle preparations. Two different models, allosteric and kinetic, have been invoked to explain these findings. The present studies investigate the mechanism of antagonism of brain 5-hydroxytryptamine (5HT)2 receptors, utilizing cells transfected with 5HT2 receptor cDNA cloned from rat brain. A proximal cellular response, phosphoinositide hydrolysis, was examined in order to minimize possible postreceptor effects. Even though LSD behaved as a partial agonist and BOL as a pure antagonist, both drugs blocked the effect of serotonin in an unsurmountable manner, i.e., increasing concentrations of serotonin could not overcome the blocking effect of LSD or BOL. Radioligand binding studies showed that preincubation of membranes with either LSD or BOL reduced the density of [3H]ketanserin binding sites, suggesting that the drugs bind tightly to the 5HT2 receptor and are not displaced during the binding assay. Two additional experiments supported this hypothesis. First, the off-rate of [3H] LSD was slow (20 min), relative to that of [3H]ketanserin (approximately 4 min). Second, when the length of incubation with [3H]ketanserin was increased to 60 min, the LSD-induced decrease in Bmax was essentially eliminated. The possibility that LSD and BOL decrease [3H]ketanserin binding by interacting with an allosteric site was rejected, because neither drug altered the rate of dissociation of [3H]ketanserin. The most parsimonious interpretation of these results is that unsurmountable antagonism reflects prolonged occupancy of the receptor by slowly reversible antagonists.

  16. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  17. The fate of P2Y-related orphan receptors: GPR80/99 and GPR91 are receptors of dicarboxylic acids.

    PubMed

    Gonzalez, Nathalie Suarez; Communi, Didier; Hannedouche, Sébastien; Boeynaems, Jean-Marie

    2004-12-01

    Several orphan G protein-coupled receptors are structurally close to the family of P2Y nucleotide receptors: GPR80/99 and GPR91 are close to P2Y(1/2/4/6/11) receptors, whereas GPR87, H963 and GPR34 are close to P2Y(12/13/14). Over the years, several laboratories have attempted without success to identify the ligands of those receptors. In early 2004, two papers have been published: One claiming that GPR80/99 is an AMP receptor, called P2Y(15), and the other one showing that GPR80/99 is a receptor for alpha-ketoglutarate, while GPR91 is a succinate receptor. The accompanying paper by Qi et al. entirely supports that GPR80/99 is an alpha-ketoglutarate receptor and not an AMP receptor. The closeness of dicarboxylic acid and P2Y nucleotide receptors might be linked to the negative charges of both types of ligands and the involvement of conserved Arg residues in their neutralization. PMID:18404396

  18. Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor

    NASA Astrophysics Data System (ADS)

    Inagaki, Takeshi; Moschetta, Antonio; Lee, Youn-Kyoung; Peng, Li; Zhao, Guixiang; Downes, Michael; Yu, Ruth T.; Shelton, John M.; Richardson, James A.; Repa, Joyce J.; Mangelsdorf, David J.; Kliewer, Steven A.

    2006-03-01

    Obstruction of bile flow results in bacterial proliferation and mucosal injury in the small intestine that can lead to the translocation of bacteria across the epithelial barrier and systemic infection. These adverse effects of biliary obstruction can be inhibited by administration of bile acids. Here we show that the farnesoid X receptor (FXR), a nuclear receptor for bile acids, induces genes involved in enteroprotection and inhibits bacterial overgrowth and mucosal injury in ileum caused by bile duct ligation. Mice lacking FXR have increased ileal levels of bacteria and a compromised epithelial barrier. These findings reveal a central role for FXR in protecting the distal small intestine from bacterial invasion and suggest that FXR agonists may prevent epithelial deterioration and bacterial translocation in patients with impaired bile flow. bacteria | biliary obstruction | epithelial barrier | ileum

  19. Role of farnesoid X receptor and bile acids in alcoholic liver disease

    PubMed Central

    Manley, Sharon; Ding, Wenxing

    2015-01-01

    Alcoholic liver disease (ALD) is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR) and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a) and PPARα (peroxisome proliferator-activated receptor alpha) in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure. PMID:26579442

  20. Role of farnesoid X receptor and bile acids in alcoholic liver disease.

    PubMed

    Manley, Sharon; Ding, Wenxing

    2015-03-01

    Alcoholic liver disease (ALD) is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR) and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a) and PPARα (peroxisome proliferator-activated receptor alpha) in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure. PMID:26579442

  1. Retinoic acid receptor alpha mediates growth inhibition by retinoids in human colon carcinoma HT29 cells.

    PubMed

    Nicke, B; Kaiser, A; Wiedenmann, B; Riecken, E O; Rosewicz, S

    1999-08-11

    Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.

  2. Oleanolic acid acrylate elicits antidepressant-like effect mediated by 5-HT1A receptor.

    PubMed

    Fajemiroye, James O; Polepally, Prabhakar R; Chaurasiya, Narayan D; Tekwani, Babu L; Zjawiony, Jordan K; Costa, Elson A

    2015-01-01

    The development of new drugs for the treatment of depression is strategic to achieving clinical needs of patients. This study evaluates antidepressant-like effect and neural mechanisms of four oleanolic acid derivatives i.e. acrylate (D1), methacrylate (D2), methyl fumarate (D3) and ethyl fumarate (D4). All derivatives were obtained by simple one-step esterification of oleanolic acid prior to pharmacological screening in the forced swimming (FS) and open field (OF) tests. Pharmacological tools like α-methyl-p-tyrosine (AMPT, catecholamine depletor), p-chlorophenylalanine (serotonin depletor), prazosin (PRAZ, selective α1-receptor antagonist), WAY-100635 (selective serotonin 5-HT1A receptor antagonist) as well as monoamine oxidase (MAO) and functional binding assays were conducted to investigate possible neural mechanisms. In the FS test, D1 showed the most promising antidepressant-like effect without eliciting locomotor incoordination. Unlike group of mice pretreated with AMPT 100 mg/kg, PCPA 100 mg/kg or PRAZ 1 mg/kg, the effect of D1 was attenuated by WAY-100635 0.3 mg/kg pretreatment. D1 demonstrated moderate inhibition of MAO-A (IC50 = 48.848 ± 1.935 μM), potency (pEC50 = 6.1 ± 0.1) and intrinsic activity (E max = 26 ± 2.0%) on 5-HT1A receptor. In conclusion, our findings showed antidepressant-like effect of D1 and possible involvement of 5-HT1A receptor.

  3. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor

    SciTech Connect

    Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E. )

    1989-05-01

    The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 {mu}M in various T4{sup +} cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment.

  4. The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors

    PubMed Central

    Binder, Veronika; Ljubojevic, Senka; Haybaeck, Johannes; Holzer, Michael; El-Gamal, Dalia; Schicho, Rudolf; Pieske, Burkert; Heinemann, Akos; Marsche, Gunther

    2014-01-01

    Objective Elevated levels of advanced oxidation protein products (AOPPs) have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis and atherosclerosis. Recent findings revealed that AOPPs are inhibitors of the major high-density lipoprotein (HDL) receptor, scavenger receptor class B, type 1 (SR-BI). Here we investigated what oxidation induced structural alterations convert plasma albumin into an HDL-receptor inhibitor. Approach and Results Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high affinity SR-BI ligands. Protection of albumin lysine-residues prior exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin lysine-residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of HDL. Conclusion Given that several potential atheroprotective activities of HDL are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease. PMID:23493288

  5. The acidic domains of the Toc159 chloroplast preprotein receptor family are intrinsically disordered protein domains

    PubMed Central

    2009-01-01

    Background The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors. Results The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs. Conclusions Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates. PMID:20042108

  6. SIGNALLING THROUGH RETINOIC ACID RECEPTORS IN CARDIAC DEVELOPMENT: DOING THE RIGHT THINGS AT THE RIGHT TIMES

    PubMed Central

    Xavier-Neto, José; Costa, Ângela M. Sousa; Figueira, Ana Carolina M.; Caiaffa, Carlo Donato; do Amaral, Fabio Neves; Peres, Lara Maldanis Cerqueira; da Silva, Bárbara Santos Pires; Santos, Luana Nunes; Moise, Alexander R.; Castillo, Hozana Andrade

    2015-01-01

    Retinoic acid (RA) is a terpenoid that is synthesized from Vitamin A/retinol (ROL) and binds to the nuclear receptors retinoic acid receptor (RAR)/retinoid X receptor (RXR) to control multiple developmental processes in vertebrates. The available clinic and experimental data provide uncontested evidence for the pleiotropic roles of RA signalling in development of multiple embryonic structures and organs such eyes, central nervous system, gonads, lungs and heart. The development of any of these above-mentioned embryonic organ systems can be effectively utilized to showcase the many strategies utilized by RA signalling. However, it is very likely that the strategies employed to transfer RA signals during cardiac development comprise the majority of the relevant and sophisticated ways through which retinoid signals can be conveyed in a complex biological system. Here, we provide the reader with arguments indicating that RA signalling is exquisitely regulated according to specific phases of cardiac development and that RA signalling itself is one of the major regulators of the timing of cardiac morphogenesis and differentiation. We will focus on the role of signalling by RA receptors (RARs) in early phases of heart development. PMID:25134739

  7. Retinoic acid and retinoid receptors: potential chemopreventive and therapeutic role in cervical cancer.

    PubMed

    Abu, Jafaru; Batuwangala, Madu; Herbert, Karl; Symonds, Paul

    2005-09-01

    Retinoids are natural and synthetic derivatives of vitamin A, which can be obtained from animal products (milk, liver, beef, fish oils, and eggs) and vegetables (carrots, mangos, sweet potatoes, and spinach). Retinoids regulate various important cellular functions in the body through specific nuclear retinoic-acid receptors and retinoid-X receptors, which are encoded by separate genes. Retinoic-acid receptors specifically bind tretinoin and alitretinoin, whereas retinoid-X receptors bind only alitretinoin. Retinoids have long been established as crucial for several essential life processes-healthy growth, vision, maintenance of tissues, reproduction, metabolism, tissue differentiation (normal, premalignant cells, and malignant cells), haemopoiesis, bone development, spermatogenesis, embryogenesis, and overall survival. Therefore, deficiency of vitamin A can lead to various unwanted biological effects. Several experimental and epidemiological studies have shown the antiproliferative activity of retinoids and their potential use in cancer treatment and chemoprevention. Emerging clinical trials have shown the chemotherapeutic and chemopreventive potential of retinoids in cancerous and precancerous conditions of the uterine cervix. In this review, we explore the potential chemopreventive and therapeutic roles of retinoids in preinvasive and invasive cervical neoplasia.

  8. Synthesis and evaluation of a (68)Ga labeled folic acid derivative for targeting folate receptors.

    PubMed

    Jain, Akanksha; Mathur, Anupam; Pandey, Usha; Bhatt, Jyotsna; Mukherjee, Archana; Ram, Ramu; Sarma, Haladhar Dev; Dash, Ashutosh

    2016-10-01

    Present work evaluates the potential of a newly synthesized (68)Ga-NOTA-folic acid conjugate for PET imaging of tumors over-expressing folate receptors (FRs). NOTA-folic acid conjugate was synthesized and characterized. It was radiolabeled with (68)Ga in ≥ 95% radiolabeling yields. In vitro cell binding studies showed a maximum cell uptake of 1.7±0.4% per million KB cells which was completely blocked on addition of cold folic acid showing specificity towards the FRs. However, further studies in tumor xenografts are warranted in order to assess the potential of (68)Ga-folic acid complex for imaging tumors over-expressing FRs. PMID:27501138

  9. The evolution of bat nucleic acid-sensing Toll-like receptors.

    PubMed

    Escalera-Zamudio, Marina; Zepeda-Mendoza, M Lisandra; Loza-Rubio, Elizabeth; Rojas-Anaya, Edith; Méndez-Ojeda, Maria L; Arias, Carlos F; Greenwood, Alex D

    2015-12-01

    We characterized the nucleic acid-sensing Toll-like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid-sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand-binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand-binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long-term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order-specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general. PMID:26503258

  10. Autoradiographic localization and characterization of [125I]lysergic acid diethylamide binding to serotonin receptors in Aplysia.

    PubMed

    Kadan, M J; Hartig, P R

    1988-03-01

    The sensitive serotonergic radioligand 2-[125I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [125I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (Kd = 0.41 nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [125I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [3H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [125I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein. Images of serotonin receptor distribution in the Aplysia nervous system were obtained from autoradiograms of [125I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [125I]lysergic acid diethylamide. These studies indicate that [125I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor

  11. Agrochemical control of plant water use using engineered abscisic acid receptors.

    PubMed

    Park, Sang-Youl; Peterson, Francis C; Mosquna, Assaf; Yao, Jin; Volkman, Brian F; Cutler, Sean R

    2015-04-23

    Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement. PMID:25652827

  12. Abscisic Acid Analogues That Act as Universal or Selective Antagonists of Phytohormone Receptors.

    PubMed

    Rajagopalan, Nandhakishore; Nelson, Ken M; Douglas, Amy F; Jheengut, Vishal; Alarcon, Idralyn Q; McKenna, Sean A; Surpin, Marci; Loewen, Michele C; Abrams, Suzanne R

    2016-09-13

    The plant hormone abscisic acid (ABA) plays many important roles in controlling plant development and physiology, from flowering to senescence. ABA is now known to exert its effects through a family of soluble ABA receptors, which in Arabidopsis thaliana has 13 members divided into three clades. Homologues of these receptors are present in other plants, also in relatively large numbers. Investigation of the roles of each homologue in mediating the diverse physiological roles of ABA is hampered by this genetic redundancy. We report herein the in vitro screening of a targeted ABA-like analogue library and identification of novel antagonist hits, including the analogue PBI686 that had been developed previously as a probe for identifying ABA-binding proteins. Further in vitro characterization of PBI686 and development of second-generation leads yielded both receptor-selective and universal antagonist hits. In planta assays in different species have demonstrated that these antagonist leads can overcome various ABA-induced physiological changes. While the general antagonists open up a hitherto unexplored avenue for controlling plant growth through inhibition of ABA-regulated physiological processes, the receptor-selective antagonist can be developed into chemical probes to explore the physiological roles of individual receptors.

  13. Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes.

    PubMed Central

    Tomic, M; Jiang, C K; Epstein, H S; Freedberg, I M; Samuels, H H; Blumenberg, M

    1990-01-01

    In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters. Images PMID:1712634

  14. Abscisic Acid Analogues That Act as Universal or Selective Antagonists of Phytohormone Receptors.

    PubMed

    Rajagopalan, Nandhakishore; Nelson, Ken M; Douglas, Amy F; Jheengut, Vishal; Alarcon, Idralyn Q; McKenna, Sean A; Surpin, Marci; Loewen, Michele C; Abrams, Suzanne R

    2016-09-13

    The plant hormone abscisic acid (ABA) plays many important roles in controlling plant development and physiology, from flowering to senescence. ABA is now known to exert its effects through a family of soluble ABA receptors, which in Arabidopsis thaliana has 13 members divided into three clades. Homologues of these receptors are present in other plants, also in relatively large numbers. Investigation of the roles of each homologue in mediating the diverse physiological roles of ABA is hampered by this genetic redundancy. We report herein the in vitro screening of a targeted ABA-like analogue library and identification of novel antagonist hits, including the analogue PBI686 that had been developed previously as a probe for identifying ABA-binding proteins. Further in vitro characterization of PBI686 and development of second-generation leads yielded both receptor-selective and universal antagonist hits. In planta assays in different species have demonstrated that these antagonist leads can overcome various ABA-induced physiological changes. While the general antagonists open up a hitherto unexplored avenue for controlling plant growth through inhibition of ABA-regulated physiological processes, the receptor-selective antagonist can be developed into chemical probes to explore the physiological roles of individual receptors. PMID:27523384

  15. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    SciTech Connect

    Melcher, Karsten; Ng, Ley-Moy; Zhou, X Edward; Soon, Fen-Fen; Xu, Yong; Suino-Powell, Kelly M; Park, Sang-Youl; Weiner, Joshua J; Fujii, Hiroaki; Chinnusamy, Viswanathan; Kovach, Amanda; Li, Jun; Wang, Yonghong; Li, Jiayang; Peterson, Francis C; Jensen, Davin R; Yong, Eu-Leong; Volkman, Brian F; Cutler, Sean R; Zhu, Jian-Kang; Xu, H Eric

    2010-01-12

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved β-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling.

  16. Aldose Reductase acts as a Selective Derepressor of PPARγ and Retinoic Acid Receptor

    PubMed Central

    Thiagarajan, Devi; Ananthakrishnan, Radha; Zhang, Jinghua; O’Shea, Karen M.; Quadri, Nosirudeen; Li, Qing; Sas, Kelli; Jing, Xiao; Rosario, Rosa; Pennathur, Subramaniam; Schmidt, Ann Marie; Ramasamy, Ravichandran

    2016-01-01

    Summary Histone deacetylase 3 (HDAC3), a chromatin modifying enzyme, requires association with the deacetylase containing domain (DAD) of the nuclear receptor co-repressors NCOR1 and SMRT for its stability and activity. Here we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically de-represses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent de-repression of PPARγ and RAR. PMID:27052179

  17. The nuclear receptor PPARγ individually responds to serotonin- and fatty acid-metabolites

    PubMed Central

    Waku, Tsuyoshi; Shiraki, Takuma; Oyama, Takuji; Maebara, Kanako; Nakamori, Rinna; Morikawa, Kosuke

    2010-01-01

    The nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPARγ through conformational changes of the Ω loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARγ to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARγ antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARγ-target gene. Collectively, these results suggest a novel agonism, in which PPARγ functions as a multiple sensor in response to distinct metabolites. PMID:20717101

  18. Dopamine D2High receptors stimulated by phencyclidines, lysergic acid diethylamide, salvinorin A, and modafinil.

    PubMed

    Seeman, Philip; Guan, Hong-Chang; Hirbec, Hélène

    2009-08-01

    Although it is commonly stated that phencyclidine is an antagonist at ionotropic glutamate receptors, there has been little measure of its potency on other receptors in brain tissue. Although we previously reported that phencyclidine stimulated cloned-dopamine D2Long and D2Short receptors, others reported that phencyclidine did not stimulate D2 receptors in homogenates of rat brain striatum. This study, therefore, examined whether phencyclidine and other hallucinogens and psychostimulants could stimulate the incorporation of [(35)S]GTP-gamma-S into D2 receptors in homogenates of rat brain striatum, using the same conditions as previously used to study the cloned D2 receptors. Using 10 microM dopamine to define 100% stimulation, phencyclidine elicited a maximum incorporation of 46% in rat striata, with a half-maximum concentration of 70 nM for phencyclidine, when compared with 80 nM for dopamine, 89 nM for salvinorin A (48 nM for D2Long), 105 nM for lysergic acid diethylamide (LSD), 120 nM for R-modafinil, 710 nM for dizocilpine, 1030 nM for ketamine, and >10,000 nM for S-modafinil. These compounds also inhibited the binding of the D2-selective ligand [(3)H]domperidone. The incorporation was inhibited by the presence of 200 microM guanylylimidodiphosphate and also by D2 blockade, using 10 microM S-sulpiride, but not by D1 blockade with 10 microM SCH23390. Hypertonic buffer containing 150 mM NaCl inhibited the stimulation by phencyclidine, which may explain negative results by others. It is concluded that phencyclidine and other psychostimulants and hallucinogens can stimulate dopamine D2 receptors at concentrations related to their behavioral actions.

  19. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    NASA Astrophysics Data System (ADS)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-11-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  20. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    PubMed Central

    Di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-01-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation. PMID:26567894

  1. Benzodiazepine receptor agonists affect both binding and gating of recombinant alpha1beta2gamma2 gamma-aminobutyric acid-A receptors.

    PubMed

    Mercik, Katarzyna; Piast, Michał; Mozrzymas, Jerzy W

    2007-05-28

    Benzodiazepines are known to act by enhancing the effect of gamma-aminobutyric acid-A receptor agonists. Positive modulation by benzodiazepines is typically ascribed to upregulation of agonist binding affinity but their effect on gamma-aminobutyric acid-A receptor gating remain unclear. In this work, we have used the ultrafast application system to examine the impact of flurazepam and zolpidem on recombinant alpha1beta2gamma2 gamma-aminobutyric acid-A receptors. As expected, both drugs strongly enhanced currents evoked by low [gamma-aminobutyric acid]. These compounds, however, also affected currents elicited by saturating agonist concentration. In particular, flurazepam and zolpidem reduced current amplitudes and slowed down the recovery process in paired-pulse experiments. Moreover, flurazepam accelerated the current rise time and zolpidem enhanced the rate and extent of desensitization. We propose that flurazepam and zolpidem modulate gamma-aminobutyric acid-A receptors by strong enhancement of agonist binding with a superimposed limited effect on the receptor gating.

  2. Essential role for retinoic acid in the promotion of CD4+ T cell effector responses via retinoic acid receptor alpha

    PubMed Central

    Hall, J.A.; Cannons, J.L.; Grainger, J.R.; Santos, L.M. Dos; Hand, T.W.; Naik, S.; Wohlfert, E.A.; Chou, D.B.; Oldenhove, G.; Robinson, M.; Grigg, M.E.; Kastenmayer, R.; Schwartzberg, P.L.; Belkaid, Y.

    2012-01-01

    SUMMARY Vitamin A and its metabolite, retinoic acid (RA), have recently been implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we show that RA is also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) is the critical mediator of these effects. Strikingly, antagonism of RAR signaling and deficiency in RARα(Rara−/−) results in a cell autonomous CD4+ T cell activation defect. Altogether, these findings reveal a fundamental role for the RA/RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses. PMID:21419664

  3. Design, synthesis and biological activity of phenoxyacetic acid derivatives as novel free fatty acid receptor 1 agonists.

    PubMed

    Li, Zheng; Wang, Xuekun; Xu, Xue; Yang, Jianyong; Xia, Wenting; Zhou, Xianhao; Huang, Wenlong; Qian, Hai

    2015-11-15

    The free fatty acid receptor 1 (FFA1) is a novel antidiabetic target for the treatment of type 2 diabetes based on particular mechanism in amplifying glucose-stimulated insulin secretion. We have previously identified a series of phenoxyacetic acid derivatives. Herein, we describe the further chemical modification of this series directed by ligand efficiency and ligand lipophilicity efficiency. All of these efforts lead to the discovery of the promising candidate 16, an excellent FFA1 agonist with robust agonistic activity (43.6 nM), desired LE and LLE values. Moreover, compound 16 revealed a great potential for improving the hyperglycemia levels in both normal and type 2 diabetic mice without the risk of hypoglycemia even at the high dose of 40 mg/kg. PMID:26482570

  4. Modulation of GABA receptors expressed in Xenopus oocytes by 13-L-hydroxylinoleic acid and food additives.

    PubMed

    Aoshima, H; Tenpaku, Y

    1997-12-01

    To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on gamma-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 microM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

  5. PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

    PubMed Central

    Chachisvilis, Mirianas

    2012-01-01

    The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone. PMID:23300710

  6. Histamine (H3) receptors modulate the excitatory amino acid receptor response of the vestibular afferents.

    PubMed

    Chávez, Hortencia; Vega, Rosario; Soto, Enrique

    2005-12-01

    Although the effectiveness of histamine-related drugs in the treatment of peripheral and central vestibular disorders may be explained by their action on the vestibular nuclei, it has also been shown that antivertigo effects can take place at the peripheral level. In this work, we examined the actions of H3 histaminergic agonists and antagonists on the afferent neuron electrical discharge in the isolated inner ear of the axolotl. Our results indicate that H3 antagonists such as thioperamide, clobenpropit, and betahistine (BH) decreased the electrical discharge of afferent neurons by interfering with the postsynaptic response to excitatory amino acid agonists. These results lend further support to the idea that the antivertigo action of histamine-related drugs may be caused, at least in part, by a decrease in the sensory input from the vestibular endorgans. The present data show that the inhibitory action of the afferent neurons discharge previously described for BH is not restricted to this molecule but is also shared by other H3 antagonists.

  7. A Comparison of the Roles of Peroxisome Proliferator-Activated Receptor and Retinoic Acid Receptor on CYP26 Regulation

    PubMed Central

    Tay, Suzanne; Dickmann, Leslie; Dixit, Vaishali

    2010-01-01

    The cytochrome P450 26 family is believed to be responsible for all-trans-retinoic acid (atRA) metabolism and elimination in the human fetus and adults. CYP26A1 and CYP26B1 mRNA is expressed in a tissue-specific manner, and mice in which the CPY26 isoform has been knocked out show distinct malformations and lethality. The aim of this study was to determine differences in CYP26A1 and CYP26B1 regulation and expression. Analysis of CYP26A1 and CYP26B1 expression in a panel of 57 human livers showed CYP26A1 to be the major CYP26 isoform present in the liver, and its expression to be subject to large interindividual variability between donors. CYP26A1 and retinoic acid receptor (RAR) β were found to be greatly inducible by atRA in HepG2 cells, whereas CYP26B1, RARα, and RARγ were induced to a much lesser extent. Based on treatments with RAR isoform-selective ligands, RARα is the major isoform responsible for CYP26A1 and RARβ induction in HepG2 cells. Classic cytochrome P450 inducers did not affect CYP26 transcription, whereas the peroxisome proliferator-activated receptor (PPAR) γ agonists pioglitazone and rosiglitazone up-regulated CYP26B1 transcription by as much as 209- ± 80-fold and CYP26A1 by 10-fold. RARβ was also up-regulated by pioglitazone and rosiglitazone. CYP26B1 induction by PPARγ agonists was abolished by the irreversible PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662), whereas RARβ and CYP26A1 induction was unaffected by GW9662. Overall, the results of this study suggest that CYP26B1 and CYP26A1 are regulated by different nuclear receptors, resulting in tissue-specific expression patterns. The fact that drugs can alter the expression of CYP26 enzymes may have toxicological and therapeutic importance. PMID:19884280

  8. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  9. Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid.

    PubMed

    Joo, Yeon Ah; Chung, Hyunjin; Yoon, Sohyun; Park, Jong Il; Lee, Ji Eun; Myung, Cheol Hwan; Hwang, Jae Sung

    2016-09-01

    Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH₂ and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca²⁺ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH₂ and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH₂-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH₂ downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH₂ in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis. PMID:27169822

  10. Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha.

    PubMed

    Hansen, Mariann F; Greibe, Eva; Skovbjerg, Signe; Rohde, Sarah; Kristensen, Anders C M; Jensen, Trine R; Stentoft, Charlotte; Kjær, Karina H; Kronborg, Camilla S; Martensen, Pia M

    2015-07-01

    The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells. We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR. The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

  11. Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid

    PubMed Central

    Joo, Yeon Ah; Chung, Hyunjin; Yoon, Sohyun; Park, Jong Il; Lee, Ji Eun; Myung, Cheol Hwan; Hwang, Jae Sung

    2016-01-01

    Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca2+ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis. PMID:27169822

  12. Amino acid sequence coevolution in the insect bursicon ligand-receptor system.

    PubMed

    Hughes, Austin L

    2012-06-01

    The pattern of amino acid residue replacement in the components of the bursicon signaling system (involving the BURSα/BURSβ heterodimer and its receptor BURSrec) was reconstructed across a phylogeny of 17 insect species, in order to test for the co-occurrence of replacements at sets of individual sites. Sets of three or more branches with perfectly concordant changes occurred to a greater extent than expected by chance, given the observed level of amino acid change. The latter sites (SPC sites) were found to have distinctive characteristics: (1) the mean number of changes was significantly lower at SPC sites than that at other sites with multiple changes; (2) SPC sites had a significantly greater tendency toward parallel amino acid changes than other sites with multiple changes, but no greater tendency toward convergent changes; and (3) parallel changes tended to involve relatively similar amino acids, as indicated by relatively low mean chemical distances. The results implicated functional constraint, permitting only a limited subset of amino acids in a given site, as a major factor in causing both parallel amino acid replacement and coordinated amino acid changes in different sites of the same protein and of interacting proteins in this system.

  13. Role of the retinoic acid receptor-α in HIV-associated nephropathy.

    PubMed

    Ratnam, Krishna K; Feng, Xiaobei; Chuang, Peter Y; Verma, Vikram; Lu, Ting-Chi; Wang, Jinshan; Jin, Yuanmeng; Farias, Eduardo F; Napoli, Joseph L; Chen, Nan; Kaufman, Lewis; Takano, Tomoko; D'Agati, Vivette D; Klotman, Paul E; He, John C

    2011-03-01

    All-trans retinoic acid protects against the development of HIV-associated nephropathy (HIVAN) in HIV-1 transgenic mice (Tg26). In vitro, all-trans retinoic acid inhibits HIV-induced podocyte proliferation and restores podocyte differentiation markers by activating its receptor-α (RARα). Here, we report that Am580, a water-soluble RARα-specific agonist, attenuated proteinuria, glomerosclerosis, and podocyte proliferation, and restored podocyte differentiation markers in kidneys of Tg26 mice. Furthermore, RARα-/- Tg26 mice developed more severe kidney and podocyte injury than did RARα+/- Tg26 mice. Am580 failed to ameliorate kidney injury in RARα-/- Tg26 mice, confirming our hypothesis that Am580 acts through RARα. Although the expression of RARα-target genes was suppressed in the kidneys of Tg26 mice and of patients with HIVAN, the expression of RARα in the kidney was not different between patients with HIVAN and minimal change disease. However, the tissue levels of retinoic acid were reduced in the kidney cortex and isolated glomeruli of Tg26 mice. Consistent with this, the expression of two key enzymes in the retinoic acid synthetic pathway, retinol dehydrogenase type 1 and 9, and the overall enzymatic activity for retinoic acid synthesis were significantly reduced in the glomeruli of Tg26 mice. Thus, a defect in the endogenous synthesis of retinoic acid contributes to loss of the protection by retinoic acid in HIVAN. Hence, RARα agonists may be potential agents for the treatment of HIVAN.

  14. Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity

    PubMed Central

    Liu, Lijing; Sonbol, Fathi-Mohamed; Huot, Bethany; Gu, Yangnan; Withers, John; Mwimba, Musoki; Yao, Jian; He, Sheng Yang; Dong, Xinnian

    2016-01-01

    It is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens. PMID:27725643

  15. Molecular basis for designing selective modulators of retinoic acid receptor transcriptional activities.

    PubMed

    Lefebvre, P

    2001-08-01

    Retinoic acid receptors are ligand-regulated transcription factors belonging to the nuclear receptor superfamily, which comprises 49 members in the human genome. all-trans retinoic acid and 9-cis retinoic acid receptors (RARs and RXRs) are each encoded by three distinct genes and several isoforms arise from alternative splicing and the use of different promoters. While RXRs are promiscuous dimerization partners of several other nuclear receptors, RARs are active, in-vivo, when associated to RXRs. Retinoids are therefore regulators of multiple physiological processes, from embryogenesis to metabolism. Different combinations of RXR:RAR heterodimers occur as a function of their tissue-specific expression and their activity is mostly conditioned by the activation status of RAR. These heterodimers are defined as non permissive heterodimers, in opposition to permissive dimers whose transcriptional activity may be modulated through RXR and its dimerization partner. The transcriptional activity of these dimers also relies on their ability to recruit nuclear coactivators and corepressors, which function as multi proteic complexes harboring several enzymatic activities (acetylases, kinases). The structure of the ligand bound to the RAR moiety of the dimer, as well as the nature of the DNA sequence to which dimers are bound, dictate the relative affinity of dimers for coactivators and thus its overall transcriptional activity. RARs are also able to repress the activity of unrelated transcription factors such as AP1 and NF-kappa-B, and therefore have potent anti proliferative and anti inflammatory properties. This review summarizes our current view of molecular mechanisms governing these various activities and emphasizes the need for a detailed understanding of how retinoids may dictate transactivating and transrepressive properties of RARs and RXRs, which may be considered as highly valuable therapeutic targets in many diseases such as cancer, skin hyperproliferation and

  16. Novel Retinoic Acid Receptor Alpha Agonists for Treatment of Kidney Disease

    PubMed Central

    Liu, Ruijie; Li, Zhengzhe; Chen, Yibang; Evans, Todd; Chuang, Peter; Das, Bhaskar; He, John Cijiang

    2011-01-01

    Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN. PMID:22125642

  17. Novel retinoic acid receptor alpha agonists for treatment of kidney disease.

    PubMed

    Zhong, Yifei; Wu, Yingwei; Liu, Ruijie; Li, Zhengzhe; Chen, Yibang; Evans, Todd; Chuang, Peter; Das, Bhaskar; He, John Cijiang

    2011-01-01

    Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

  18. Predicting G-protein-coupled receptors families using different physiochemical properties and pseudo amino acid composition.

    PubMed

    Rehman, Zia-Ur; Mirza, Muhammad Tayyeb; Khan, Asifullah; Xhaard, Henri

    2013-01-01

    G-protein-coupled receptors (GPCRs) initiate signaling pathways via trimetric guanine nucleotide-binding proteins. GPCRs are classified based on their ligand-binding properties and molecular phylogenetic analyses. Nonetheless, these later analyses are in most case dependent on multiple sequence alignments, themselves dependent on human intervention and expertise. Alignment-free classifications of GPCR sequences, in addition to being unbiased, present many applications uncovering hidden physicochemical parameters shared among specific groups of receptors, to being used in automated workflows for large-scale molecular modeling applications. Current alignment-free classification methods, however, do not reach a full accuracy. This chapter discusses how GPCRs amino acid sequences can be classified using pseudo amino acid composition and multiscale energy representation of different physiochemical properties of amino acids. A hybrid feature extraction strategy is shown to be suitable to represent GPCRs and to be able to exploit GPCR amino acid sequence discrimination capability in spatial as well as transform domain. Classification strategies such as support vector machine and probabilistic neural network are then discussed in regards to GPCRs classification. The work of GPCR-Hybrid web predictor is also discussed.

  19. PLZF is a negative regulator of retinoic acid receptor transcriptional activity

    PubMed Central

    Martin, Perrine J; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2003-01-01

    Background Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. Results We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. Conclusion Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled. PMID:14521715

  20. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

    PubMed Central

    Choi, Sun-Hye; Jung, Seok-Won; Lee, Byung-Hwan; Kim, Hyeon-Joong; Hwang, Sung-Hee; Kim, Ho-Kyoung; Nah, Seung-Yeol

    2015-01-01

    Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs), which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter, and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology. PMID:26578955

  1. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

    PubMed Central

    Izzo, Emanuela; Auta, James; Impagnatiello, Francesco; Pesold, Christine; Guidotti, Alessandro; Costa, Erminio

    2001-01-01

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72–96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABAA (γ-aminobutyric acid type A) receptor subunits (decrease in γ2 and α1; increase in α5) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD67. In contrast, dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal. PMID:11248104

  2. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

    PubMed

    Izzo, E; Auta, J; Impagnatiello, F; Pesold, C; Guidotti, A; Costa, E

    2001-03-13

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

  3. Retinoic Acid Receptor-Mediated Induction of ABCA1 in Macrophages

    PubMed Central

    Costet, Philippe; Lalanne, Florent; Gerbod-Giannone, Marie C.; Molina, Jennifer R.; Fu, Xuan; Lund, Erik G.; Gudas, Lorraine J.; Tall, Alan R.

    2003-01-01

    ABCA1, the mutant molecule in Tangier Disease, mediates efflux of cellular cholesterol to apoA-I and is induced by liver X receptor (LXR)/retinoid X receptor (RXR) transcription factors. Retinoic acid receptor (RAR) activators (all-trans-retinoic acid [ATRA] and TTNPB) were found to increase ATP-binding cassette transporter 1 (ABCA1) mRNA and protein in macrophages. In cellular cotransfection assays, RARγ/RXR activated the human ABCA1 promoter, via the same direct repeat 4 (DR4) promoter element as LXR/RXR. Chromatin immunoprecipitation analysis in macrophages confirmed the binding of RARγ/RXR to the ABCA1 promoter DR4 element in the presence of ATRA, with weaker binding of RARα/RXR, and no binding of RARβ/RXR. However, in macrophages from RARγ−/− mice, TTNPB still induced ABCA1, in association with marked upregulation of RARα, suggesting that high levels of RARα can compensate for the absence of RARγ. Dose-response experiments with ATRA in mouse primary macrophages showed that other LXR target genes were weakly induced (ABCG1 and SREBP-1c) or not induced (apoE and LXRα). The more specific RAR activator TTNPB did not induce SREBP-1c in mouse primary macrophages or liver. These studies indicate a direct role of RARγ/RXR in induction of macrophage ABCA1. PMID:14560020

  4. Functional Analysis of Free Fatty Acid Receptor GPR120 in Human Eosinophils: Implications in Metabolic Homeostasis

    PubMed Central

    Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

    2015-01-01

    Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system. PMID:25790291

  5. Recognition and sequestration of ω-fatty acids by a cavitand receptor

    PubMed Central

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-01-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring “prestacked” aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules—unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide—from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules. PMID:26305974

  6. Recognition and sequestration of ω-fatty acids by a cavitand receptor.

    PubMed

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-09-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring "prestacked" aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules-unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide-from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules.

  7. Comparison of the effects of pelargonic acid vanillylamide and capsaicin on human vanilloid receptors.

    PubMed

    Weiser, Thomas; Roufogalis, Basil; Chrubasik, Sigrun

    2013-07-01

    Pelargonic acid vanillylamide is like capsaicin a natural capsaicinoid from chili peppers and commonly used in food additives to create a hot sensation, even in self-defense pepper sprays and as an alternative to capsaicin in medical products for topical treatment of pain. Although the chemical structures of both compounds are similar, preclinical data suggest that capsaicin is the more potent compound. We therefore performed voltage-clamp recordings using cells transfected with the human vanilloid receptor TRPV1 in order to assess the responses of pelargonic acid vanillylamide and capsaicin at the receptor level. We provide evidence that at the molecular target TRPV1, the concentration-response curves, kinetics of current activation, as well as inhibition by the competitive antagonist capsazepine were not significantly different between the two capsaicinoids. We suggest that the different effects of the two capsaicinoids observed in previous studies may rather be due to different physicochemical or pharmacokinetic properties than to different pharmacological profiles at the receptor level. PMID:22961689

  8. Comparison of the effects of pelargonic acid vanillylamide and capsaicin on human vanilloid receptors.

    PubMed

    Weiser, Thomas; Roufogalis, Basil; Chrubasik, Sigrun

    2013-07-01

    Pelargonic acid vanillylamide is like capsaicin a natural capsaicinoid from chili peppers and commonly used in food additives to create a hot sensation, even in self-defense pepper sprays and as an alternative to capsaicin in medical products for topical treatment of pain. Although the chemical structures of both compounds are similar, preclinical data suggest that capsaicin is the more potent compound. We therefore performed voltage-clamp recordings using cells transfected with the human vanilloid receptor TRPV1 in order to assess the responses of pelargonic acid vanillylamide and capsaicin at the receptor level. We provide evidence that at the molecular target TRPV1, the concentration-response curves, kinetics of current activation, as well as inhibition by the competitive antagonist capsazepine were not significantly different between the two capsaicinoids. We suggest that the different effects of the two capsaicinoids observed in previous studies may rather be due to different physicochemical or pharmacokinetic properties than to different pharmacological profiles at the receptor level.

  9. Expression and distribution of sialic acid influenza virus receptors in wild birds

    PubMed Central

    França, M.; Stallknecht, D. E.; Howerth, E. W.

    2013-01-01

    Avian influenza (AI) viruses have been detected in more than 105 wild bird species from 12 different orders but species-related differences in susceptibility to AI viruses exist. Expression of α2,3-linked (avian-type) and α2,6linked (human type) sialic acid (SA) influenza virus receptors in tissues is considered to be one of the determinants of the host range and tissue tropism of influenza viruses. We investigated the expression of these SA receptors in 37 wild bird species from 11 different orders by lectin histochemistry. Two isoforms of Maackia amurensis (MAA) lectin, MAA1 and MAA2, were used to detect α2,3-linked SA and Sambucus nigra (SNA) lectin was used to detect α2,6-linked SA. All species evaluated expressed α2,3-linked and α2,6-linked SA receptors in endothelial cells and renal tubular epithelial cells. Both α2,3-linked and α-2,6-linked SA receptors were expressed in respiratory and intestinal tract tissues of aquatic and terrestrial wild bird species from different taxa, but differences in SA expression and in the predominant isoform of MAA lectin bound were observed. With a few possible exceptions, these observed differences were not generally predictive of reported species susceptibility to AI viruses based on published experimental and field data. PMID:23391183

  10. Effects of perfluoroalkyl acids on the function of the thyroid hormone and the aryl hydrocarbon receptor.

    PubMed

    Long, Manhai; Ghisari, Mandana; Bonefeld-Jørgensen, Eva Cecilie

    2013-11-01

    Perfluoroalkyl acids (PFAAs) are perfluorinated compounds that widely exist in the environment and can elicit adverse effects including endocrine disruption in humans and animals. This study investigated the effect of seven PFAAs on the thyroid hormone (TH) system assessing the proliferation of the 3,3',5-triiodo-L-thryonine (T3)-dependent rat pituitary GH3 cells using the T-screen assay and the effect on the aryl hydrocarbon receptor (AhR) transactivation in the AhR-luciferase reporter gene bioassay. A dose-dependent impact on GH3 cells was observed in the range 1×10(-9)-1×10(-4) M: seven PFAAs (perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), and perfluorododecanoic acid (PFDoA)) inhibited the GH3 cell growth, and four PFAAs (PFOS, PFHxS, PFNA, and PFUnA) antagonized the T3-induced GH3 cell proliferation. At the highest test concentration, PFHxS showed a further increase of the T3-induced GH3 growth. Among the seven tested PFAAs, only PFDoA and PFDA elicited an activating effect on the AhR. In conclusion, PFAAs possess in vitro endocrine-disrupting potential by interfering with TH and AhR functions, which need to be taken into consideration when assessing the impact on human health. PMID:23539207

  11. Mapping amino acids of the measles virus hemagglutinin responsible for receptor (CD46) downregulation.

    PubMed

    Bartz, R; Brinckmann, U; Dunster, L M; Rima, B; Ter Meulen, V; Schneider-Schaulies, J

    1996-10-01

    We compared the amino acid sequences of groups of receptor (CD46) downregulating and nondownregulating measles virus (MV) hemagglutinins (Hs) and identified seven group-specific differences as candidates for the mediation of the observed differential effects. Using site-directed mutagenesis, we mutated the chosen amino acids of the H of MV-strain WTF (WTF-H), a nondownregulating H, and Introduced the corresponding amino acids of Edmonston-H (Edm-H), a downregulating H. We identified four amino acids, 211G, 243R, 451V, and 481Y, which influenced the downregulative function when introduced into WTF-H. The double mutation 451V and 481Y in WTF-H led to a degree of CD46 downregulation comparable to that of Edm-H. Conversely, introducing amino acids 451E and 481N into Edm-H resulted in a loss of the downregulative function. These results indicate that these amino acids play a decisive role in the H-CD46 interaction. PMID:8862431

  12. Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

    PubMed

    Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

    2014-03-01

    Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression.

  13. Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity

    PubMed Central

    Mueller, Michaela; Thorell, Anders; Claudel, Thierry; Jha, Pooja; Koefeler, Harald; Lackner, Carolin; Hoesel, Bastian; Fauler, Guenter; Stojakovic, Tatjana; Einarsson, Curt; Marschall, Hanns-Ulrich; Trauner, Michael

    2015-01-01

    Background & Aims Bile acids (BAs) are major regulators of hepatic BA and lipid metabolism but their mechanisms of action in non-alcoholic fatty liver disease (NAFLD) are still poorly understood. Here we aimed to explore the molecular and biochemical mechanisms of ursodeoxycholic acid (UDCA) in modulating the cross-talk between liver and visceral white adipose tissue (vWAT) regarding BA and cholesterol metabolism and fatty acid/lipid partitioning in morbidly obese NAFLD patients. Methods In this randomized controlled pharmacodynamic study, we analyzed serum, liver and vWAT samples from 40 well-matched morbidly obese patients receiving UDCA (20 mg/kg/day) or no treatment three weeks prior to bariatric surgery. Results Short term UDCA administration stimulated BA synthesis by reducing circulating fibroblast growth factor 19 and farnesoid X receptor (FXR) activation, resulting in cholesterol 7α-hydroxylase induction mirrored by elevated C4 and 7α-hydroxycholesterol. Enhanced BA formation depleted hepatic and LDL-cholesterol with subsequent activation of the key enzyme of cholesterol synthesis 3-hydroxy-3-methylglutaryl-CoA reductase. Blunted FXR anti-lipogenic effects induced lipogenic stearoyl-CoA desaturase (SCD) in the liver, thereby increasing hepatic triglyceride content. In addition, induced SCD activity in vWAT shifted vWAT lipid metabolism towards generation of less toxic and more lipogenic monounsaturated fatty acids such as oleic acid. Conclusion These data demonstrate that by exerting FXR-antagonistic effects, UDCA treatment in NAFLD patients strongly impacts on cholesterol and BA synthesis and induces neutral lipid accumulation in both liver and vWAT. PMID:25617503

  14. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  15. Dissecting the Role of Retinoic Acid Receptor Isoforms in the CD8 Response to Infection

    PubMed Central

    Guo, Yanxia; Lee, Yu-Chi; Brown, Chrysothemis; Zhang, Weijun; Usherwood, Edward; Noelle, Randolph J.

    2015-01-01

    Vitamin A deficiency leads to increased susceptibility to a spectrum of infectious diseases. The studies presented dissect the intrinsic role of each of the retinoic acid receptor (RAR) isoforms in the clonal expansion, differentiation, and survival of pathogen-specific CD8 T cells in vivo. The data show that RARα is required for the expression of gut-homing receptors on CD8+ T cells and survival of CD8+ T cells in vitro. Furthermore, RARα is essential for survival of CD8+ T cells in vivo following Listeria monocytogenes infection. In contrast, RARβ deletion leads to modest deficiency in Ag-specific CD8+ T cell expansion during infection. The defective survival of RARα-deficient CD8+ T cells leads to a deficiency in control of L. monocytogenes expansion in the spleen. To our knowledge, these are the first comparative studies of the role of RAR isoforms in CD8+ T cell immunity. PMID:24610012

  16. Bile acid transporters and regulatory nuclear receptors in the liver and beyond

    PubMed Central

    Halilbasic, Emina; Claudel, Thierry; Trauner, Michael

    2013-01-01

    Summary Bile acid (BA) transporters are critical for maintenance of the enterohepatic BA circulation where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization and excretion of cholesterol, as well as antimicrobial and metabolic effects. Tight regulation of BA transporters via nuclear receptors is necessary to maintain proper BA homeostasis. Hereditary and acquired defects of BA transporters are involved in the pathogenesis of several hepatobiliary disorders including cholestasis, gallstones, fatty liver disease and liver cancer, but also play a role in intestinal and metabolic disorders beyond the liver. Thus, pharmacological modification of BA transporters and their regulatory nuclear receptors opens novel treatment strategies for a wide range of disorders. PMID:22885388

  17. G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

    PubMed Central

    Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; Booz, Valeria; Bruzzone, Santina; Sturla, Laura; Di Bona, Melody; Pesce, Mattia; Usai, Cesare; De Flora, Antonio; Zocchi, Elena

    2016-01-01

    Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation. PMID:27222287

  18. Inhibitory effects of lysophosphatidic acid receptor-5 on cellular functions of sarcoma cells.

    PubMed

    Araki, Mutsumi; Kitayoshi, Misaho; Dong, Yan; Hirane, Miku; Ozaki, Shuhei; Mori, Shiori; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

    2014-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). Here, we investigated the effects of LPA signaling via LPA5 on cellular functions of sarcoma cells by generating Lpar5 overexpressing and Lpar5 knockdown cells from rat osteosarcoma and malignant fibrous histiocytoma cells, respectively. The cell motility activity of Lpar5 overexpressing cells was significantly lower, while Lpar5 knockdown cells showed high cell motility, compared with respective controls. Gelatin zymography showed that LPA5 suppressed the activation of matrix metalloproteinase-2. LPA5 also inhibited the cell motility activity of endothelial cells, correlating with the expression levels of vascular endothelial growth factor genes. These results suggest that LPA signaling via LPA5 negatively regulates the cellular functions of rat sarcoma cells. PMID:24798396

  19. Nipecotic acid ethyl ester: a cholinergic agonist that may differentiate muscarinic receptor subtypes

    SciTech Connect

    Zorn, S.H.; Duman, R.S.; Enna, S.J.; Krogsgaard-Larsen, P.; Micheletti, R.; Giraldo, E.; Giachetti, A.

    1986-03-05

    Reports indicate that nipecotic acid ethyl ester (NAEE) displays cholinomimetic properties in vivo. In the present study a series of physiological and biochemical tests were conducted to characterize this action. NAEE had a negative inotropic effect on the guinea pig atrium, and stimulated contraction of the guinea pig ileum and isolated mouse stomach strip at concentrations similar to bethanechol (BCH). The atrial and ilial effects were reversed by atropine. Unlike BCH, NAEE had no effect on basal acid secretion in the isolated mouse stomach at concentrations < 100 ..mu..M. NAEE was more potent than carbachol (CCH) in displacing /sup 3/H-ONB binding from rat brain membranes. The potency of NAEE to inhibit antagonist binding in rat heart membranes was enhanced by Mg/sup + +/ (Hill coefficient < 1.0) and reduced by Gpp(NH)p. Like CCH, NAEE inhibited GTP-stimulated adenylate cyclase in rat brain striatal membranes. As compared to CCH, NAEE had little effect (< 5%) as a stimulator of inositol phosphate (IP) production in rat brain slices. The results indicate that NAEE is a direct-acting muscarinic receptor agonist. Moreover, its differential effects on acid secretion, IP accumulation, and adenylate cyclase suggest that it may be useful for defining cholinergic receptor subclasses.

  20. Membrane bile acid receptor TGR5 predicts good prognosis in ampullary adenocarcinoma patients with hyperbilirubinemia

    PubMed Central

    Chen, Min-Chan; Chen, Yi-Ling; Wang, Tzu-Wen; Hsu, Hui-Ping; Lai, Ming-Derg

    2016-01-01

    Bile acids are potential carcinogens in gastrointestinal cancer, and interact with nuclear and membrane receptors to initiate downstream signaling. The effect of TGR5 [also known as G protein-coupled bile acid receptor 1 (GPBAR1)] on cancer progression is dependent on the tissue where it is activated. In this report, the function of TGR5 expression in cancer was studied using a bioinformatic approach. TGR5 expression in ampullary adenocarcinoma and normal duodenum was compared by western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry (IHC). High GPBAR1 gene expression was found to be an indicator of worse prognosis in gastric and breast cancer patients, and an indication of better prognosis in ovarian cancer patients. The level of GPBAR1 gene expression was higher in bile-acid exposed cancer than in other types of cancer, and was increased in well-differentiated ampullary adenocarcinoma. Negative, weak or mild expression of TGR5 was correlated with younger age, higher plasma level of total/direct bilirubin, higher plasma concentration of CA-125, advanced tumor stage and advanced AJCC TNM stage. The disease-specific survival rate was highest in ampullary adenocarcinoma patients with high TGR5 expression and high total bilirubin level. In summary, TGR5 functions as a tumor-suppressor in patients with ampullary adenocarcinoma and preoperative hyperbilirubinemia. Further study of the suppressive mechanism may provide a new therapeutic option for patients with ampullary adenocarcinoma. PMID:27510297

  1. Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors.

    PubMed

    Gududuru, Veeresa; Zeng, Kui; Tsukahara, Ryoko; Makarova, Natalia; Fujiwara, Yuko; Pigg, Kathryn R; Baker, Daniel L; Tigyi, Gabor; Miller, Duane D

    2006-01-15

    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. PMID:16290140

  2. Membrane bile acid receptor TGR5 predicts good prognosis in ampullary adenocarcinoma patients with hyperbilirubinemia.

    PubMed

    Chen, Min-Chan; Chen, Yi-Ling; Wang, Tzu-Wen; Hsu, Hui-Ping; Lai, Ming-Derg

    2016-10-01

    Bile acids are potential carcinogens in gastrointestinal cancer, and interact with nuclear and membrane receptors to initiate downstream signaling. The effect of TGR5 [also known as G protein-coupled bile acid receptor 1 (GPBAR1)] on cancer progression is dependent on the tissue where it is activated. In this report, the function of TGR5 expression in cancer was studied using a bioinformatic approach. TGR5 expression in ampullary adenocarcinoma and normal duodenum was compared by western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry (IHC). High GPBAR1 gene expression was found to be an indicator of worse prognosis in gastric and breast cancer patients, and an indication of better prognosis in ovarian cancer patients. The level of GPBAR1 gene expression was higher in bile‑acid exposed cancer than in other types of cancer, and was increased in well-differentiated ampullary adenocarcinoma. Negative, weak or mild expression of TGR5 was correlated with younger age, higher plasma level of total/direct bilirubin, higher plasma concentration of CA-125, advanced tumor stage and advanced AJCC TNM stage. The disease-specific survival rate was highest in ampullary adenocarcinoma patients with high TGR5 expression and high total bilirubin level. In summary, TGR5 functions as a tumor-suppressor in patients with ampullary adenocarcinoma and preoperative hyperbilirubinemia. Further study of the suppressive mechanism may provide a new therapeutic option for patients with ampullary adenocarcinoma. PMID:27510297

  3. Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors.

    PubMed

    Gududuru, Veeresa; Zeng, Kui; Tsukahara, Ryoko; Makarova, Natalia; Fujiwara, Yuko; Pigg, Kathryn R; Baker, Daniel L; Tigyi, Gabor; Miller, Duane D

    2006-01-15

    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.

  4. Oleanolic acid acrylate elicits antidepressant-like effect mediated by 5-HT1A receptor

    PubMed Central

    Fajemiroye, James O.; Polepally, Prabhakar R.; Chaurasiya, Narayan D.; Tekwani, Babu L.; Zjawiony, Jordan K.; Costa, Elson A.

    2015-01-01

    The development of new drugs for the treatment of depression is strategic to achieving clinical needs of patients. This study evaluates antidepressant-like effect and neural mechanisms of four oleanolic acid derivatives i.e. acrylate (D1), methacrylate (D2), methyl fumarate (D3) and ethyl fumarate (D4). All derivatives were obtained by simple one-step esterification of oleanolic acid prior to pharmacological screening in the forced swimming (FS) and open field (OF) tests. Pharmacological tools like α-methyl-p-tyrosine (AMPT, catecholamine depletor), p-chlorophenylalanine (serotonin depletor), prazosin (PRAZ, selective α1-receptor antagonist), WAY-100635 (selective serotonin 5-HT1A receptor antagonist) as well as monoamine oxidase (MAO) and functional binding assays were conducted to investigate possible neural mechanisms. In the FS test, D1 showed the most promising antidepressant-like effect without eliciting locomotor incoordination. Unlike group of mice pretreated with AMPT 100 mg/kg, PCPA 100 mg/kg or PRAZ 1 mg/kg, the effect of D1 was attenuated by WAY-100635 0.3 mg/kg pretreatment. D1 demonstrated moderate inhibition of MAO-A (IC50 = 48.848 ± 1.935 μM), potency (pEC50 = 6.1 ± 0.1) and intrinsic activity (Emax = 26 ± 2.0%) on 5-HT1A receptor. In conclusion, our findings showed antidepressant-like effect of D1 and possible involvement of 5-HT1A receptor. PMID:26199018

  5. Distinct Pathways of ERK1/2 Activation by Hydroxy-Carboxylic Acid Receptor-1

    PubMed Central

    Li, Guo; Wang, Hui-qian; Wang, Li-hui; Chen, Ru-ping; Liu, Jun-ping

    2014-01-01

    Mechanistic investigations have shown that, upon agonist activation, hydroxy-carboxylic acid receptor-1(HCA1) couples to a Gi protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for HCA1 signaling remain largely unknown. Using CHO-K1 cells stably expressing HCA1, and L6 cells, which endogenously express rat HCA1 receptors, we found that activation of ERK1/2 by HCA1 was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that HCA1 induced ERK1/2 activation via the extracellular Ca2+, PKC and IGF-I receptor transactivation-dependent pathways. In addition, we observed that pretreated the cells with M119K, an inhibitor of Gβγ subunit-dependent signaling, effectively attenuated the ERK1/2 activation triggered by HCA1, suggesting a critical role for βγ-subunits in HCA1-activated ERK1/2 phosphorylation. Furthermore, the present results also indicated that the arrestin2/3 were not required for ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to agonist, HCA1 receptors initially activate Gi, leading to dissociation of the Gβγ subunit from activated Gi, and subsequently induce ERK1/2 activation via two distinct pathways: one PKC-dependent pathway and the other IGF-IR transactivation-dependent pathway. Our results provide the first in-depth evidence that defines the molecular mechanism of HCA1-mediated ERK1/2 activation. PMID:24671202

  6. Elicidation by a H-2-receptor antagonist of the significance of mucosal histamine mobilization in exciting acid secretion.

    PubMed Central

    Lundell, L

    1975-01-01

    1. The consequence of H-2-receptor blockade for the secretory responses of the gastric mucosa to hormonal or cholinergic stimulation was studied in conscious rats with Heindenhain pouches or Pavlov pouches with the antrum retained or resected. 2. Metiamide almost completely abolished acid secretion induced by pentagastrin without altering significantly the amount of histamine excreted in the urine. Histamine mobilization on pentagastrin infusion determined in vitro, seemed to be larger during H-2-receptor blockade than with pentagastrin alone. 3. CCK-PZ mobilized mucosal histamine to a considerable extent; the secretory response to this hormone was completely abolished by H-2-receptor blockade. 4. Acid secretion in response to 2-deoxy-D-glucose was inhibited by H-2-receptor blockade in the presence or absence of the antrum; however the inhibition was less complete than with hormone-induced secretion. 5. The acid secretory response to 100 mg/kg of 2-deoxy-D-glucose appeared to be less susceptible to H-2-receptor blockade than that of 50-mg/kg of 2-deoxy-D-glucose. 6. Feeding induced a secretory response in the Pavlov pouch which initially was more effectively inhibited by H-2-receptor blockade than the response to 2-deoxy-D-glucose. In the absence of antral gastrin secretion by either stimulus was equally inhibited. 7. Methacholine-induced acid secretion was inhibited by infusion of the H-2-receptor antagonist, an inhibition that was absent when pentagastrin was concomitantly infused. 8. Although acid secretion induced by cholinergic stimuli was readily inhibited by the H-2-receptor antagonist, slight or nor inhibition was noted on pepsin secretion. 9. The role of histamine as a physiological stimulus for the parietal cell is discussed in view of the fact that the secretory effect of natural stimuli, known or demonstrated to mobilize mucosal histamine, is restrained by H-2-receptor blockade. PMID:49418

  7. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  8. Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

    PubMed

    Lee, Byung-Hwan; Kim, Jisu; Lee, Ra Mi; Choi, Sun-Hye; Kim, Hyeon-Joong; Hwang, Sung-Hee; Lee, Myung Koo; Bae, Chun-Sik; Kim, Hyoung-Chun; Rhim, Hyewon; Lim, Kiwon; Nah, Seung-Yeol

    2016-01-26

    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 β-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 β-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. PMID:26706688

  9. Arginine of retinoic acid receptor beta which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity.

    PubMed

    Zhang, Zeng Ping; Hutcheson, Juliet M; Poynton, Helen C; Gabriel, Jerome L; Soprano, Kenneth J; Soprano, Dianne Robert

    2003-01-15

    The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). Consistent with the X-ray crystal structures of RARalpha and RARgamma, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (alphaArg(276), betaArg(269), and gammaArg(278)) for coordination with the carboxyl group of RA. However, mutation of Arg(269) to Ala in RARbeta causes only a 3- to 6-fold increase in the K(d) for RA and EC(50) in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARalpha or RARgamma causes a 110-fold and a 45-fold increase in EC(50) value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of betaR269A to a mutant which mimics either RARalpha ligand selectivity (betaA225S/R269A) or RARgamma ligand selectivity (betaI263M/R269A/V338A). Our results demonstrate that in RARbeta mutants that acquire either RARalpha or RARgamma ligand specificity the Arg(269) position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARbeta. Furthermore, three mutant receptors (betaA225S/R269A, betaA225S/F279, and alphaF286A) were found to have a greater than wild-type affinity for the RARalpha-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARbeta and the X-ray crystal structures of the LBD of both RARalpha and RARgamma are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg(269) in RARbeta compared to its homologous Arg in RARalpha and RARgamma.

  10. Recessive and dominant mutations in retinoic acid receptor beta in cases with microphthalmia and diaphragmatic hernia.

    PubMed

    Srour, Myriam; Chitayat, David; Caron, Véronique; Chassaing, Nicolas; Bitoun, Pierre; Patry, Lysanne; Cordier, Marie-Pierre; Capo-Chichi, José-Mario; Francannet, Christine; Calvas, Patrick; Ragge, Nicola; Dobrzeniecka, Sylvia; Hamdan, Fadi F; Rouleau, Guy A; Tremblay, André; Michaud, Jacques L

    2013-10-01

    Anophthalmia and/or microphthalmia, pulmonary hypoplasia, diaphragmatic hernia, and cardiac defects are the main features of PDAC syndrome. Recessive mutations in STRA6, encoding a membrane receptor for the retinol-binding protein, have been identified in some cases with PDAC syndrome, although many cases have remained unexplained. Using whole-exome sequencing, we found that two PDAC-syndrome-affected siblings, but not their unaffected sibling, were compound heterozygous for nonsense (c.355C>T [p.Arg119(∗)]) and frameshift (c.1201_1202insCT [p.Ile403Serfs(∗)15]) mutations in retinoic acid receptor beta (RARB). Transfection studies showed that p.Arg119(∗) and p.Ile403Serfs(∗)15 altered RARB had no transcriptional activity in response to ligands, confirming that the mutations induced a loss of function. We then sequenced RARB in 15 subjects with anophthalmia and/or microphthalmia and at least one other feature of PDAC syndrome. Surprisingly, three unrelated subjects with microphthalmia and diaphragmatic hernia showed de novo missense mutations affecting the same codon; two of the subjects had the c.1159C>T (Arg387Cys) mutation, whereas the other one carried the c.1159C>A (p.Arg387Ser) mutation. We found that compared to the wild-type receptor, p.Arg387Ser and p.Arg387Cys altered RARB induced a 2- to 3-fold increase in transcriptional activity in response to retinoic acid ligands, suggesting a gain-of-function mechanism. Our study thus suggests that both recessive and dominant mutations in RARB cause anophthalmia and/or microphthalmia and diaphragmatic hernia, providing further evidence of the crucial role of the retinoic acid pathway during eye development and organogenesis.

  11. The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.

    PubMed

    Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M; Angueira, Anthony R; Brodsky, Michael; Hayes, M Geoffrey; Kovatcheva-Datchary, Petia; Bäckhed, Fredrik; Gilbert, Jack A; Lowe, William L; Layden, Brian T

    2015-11-15

    The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis. PMID:26394664

  12. Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication.

    PubMed

    Kim, Yunjeong; Chang, Kyeong-Ok

    2011-12-01

    Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.

  13. Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

    PubMed Central

    Cook, Donald N.; Kang, Hong Soon; Jetten, Anton M.

    2015-01-01

    In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs). We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated. PMID:26878025

  14. Redox control of retinoic acid receptor activity: a novel mechanism for retinoic acid resistance in melanoma cells.

    PubMed

    Demary, K; Wong, L; Liou, J S; Faller, D V; Spanjaard, R A

    2001-06-01

    Retinoic acid (RA) slows growth and induces differentiation of tumor cells through activation of RA receptors (RARs). However, melanoma cell lines display highly variable responsiveness to RA, which is a poorly understood phenomenon. By using Northern and Western blot analyses, we show that RA-resistant A375 and RA-responsive S91 melanoma cells express comparable levels of major components of RAR-signaling pathways. However, A375 cells have substantially higher intracellular reactive oxygen species (ROS) levels than S91 cells. Lowering ROS levels in A375 cells through hypoxic culture conditions restores RAR-dependent trans-activity, which could be further enhanced by addition of the antioxidant N-acetyl-cysteine. Hypoxia also enhances RAR activity in the moderately RA-responsive C32 cells, which have intermediate ROS levels. Conversely, increasing oxidative stress in highly RA-responsive S91 and B16 cells, which have low ROS levels, by treatment with H(2)O(2) impairs RAR activity. Consistent with these observations, RA more potently inhibited the proliferation of hypoxic A375 cells than that of normoxic cells. Oxidative states diminish, whereas reducing conditions enhance, DNA binding of retinoid X receptor/RAR heterodimers in vitro, providing a molecular basis for the observed inverse correlation between RAR activity and ROS levels. The redox state of melanoma cells provides a novel, epigenetic control mechanism of RAR activity and RA resistance. PMID:11356710

  15. Gene Interaction Network Suggests Dioxin Induces a Significant Linkage between Aryl Hydrocarbon Receptor and Retinoic Acid Receptor Beta

    PubMed Central

    Toyoshiba, Hiroyoshi; Yamanaka, Takeharu; Sone, Hideko; Parham, Frederick M.; Walker, Nigel J.; Martinez, Jeanelle; Portier, Christopher J.

    2004-01-01

    Gene expression arrays (gene chips) have enabled researchers to roughly quantify the level of mRNA expression for a large number of genes in a single sample. Several methods have been developed for the analysis of gene array data including clustering, outlier detection, and correlation studies. Most of these analyses are aimed at a qualitative identification of what is different between two samples and/or the relationship between two genes. We propose a quantitative, statistically sound methodology for the analysis of gene regulatory networks using gene expression data sets. The method is based on Bayesian networks for direct quantification of gene expression networks. Using the gene expression changes in HPL1A lung airway epithelial cells after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin at levels of 0.1, 1.0, and 10.0 nM for 24 hr, a gene expression network was hypothesized and analyzed. The method clearly demonstrates support for the assumed network and the hypothesis linking the usual dioxin expression changes to the retinoic acid receptor system. Simulation studies demonstrated the method works well, even for small samples. PMID:15345368

  16. Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

    PubMed Central

    Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

    2004-01-01

    The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

  17. GPR109A, GPR109B and GPR81, a family of hydroxy-carboxylic acid receptors.

    PubMed

    Ahmed, Kashan; Tunaru, Sorin; Offermanns, Stefan

    2009-11-01

    G-protein-coupled receptors (GPCRs) are the most versatile receptor family as they have the ability to respond to chemically diverse ligands. Despite intensive efforts during the past two decades, there are still more than 100 orphan GPCRs for which endogenous ligands are unknown. Recently, GPR109A, GPR109B and GPR81, which form a GPCR subfamily, have been deorphanized. The physiological ligands of these receptors are the ketone body 3-hydroxy-butyrate, the metabolite 2-hydroxy-propanoate (lactate) as well as the beta-oxidation intermediate 3-hydroxy-octanoate. Thus, this receptor subfamily is activated by hydroxy-carboxylic acid ligands which are intermediates of energy metabolism. All three receptors are predominantly expressed in adipocytes and mediate antilipolytic effects. In this article, we propose that the hydroxy-carboxylic acid structure of their endogenous ligands is the defining property of this receptor subfamily and that hydroxy-carboxylic acid receptors function as metabolic sensors which fine-tune the regulation of metabolic pathways.

  18. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119

    PubMed Central

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2016-01-01

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor. PMID:24184668

  19. Omega-3 Fatty Acids and Other FFA4 Agonists Inhibit Growth Factor Signaling in Human Prostate Cancer Cells

    PubMed Central

    Liu, Ze; Hopkins, Mandi M.; Zhang, Zhihong; Quisenberry, Chrystal B.; Fix, Louise C.; Galvan, Brianna M.

    2015-01-01

    Omega-3 fatty acids (n-3 FAs) are proposed to have many beneficial effects on human health. However, the mechanisms underlying their potential cancer preventative effects are unclear. G protein–coupled receptors (GPCRs) of the free fatty acid receptor (FFAR) family, FFA1/GPR40 and FFA4/GPR120, specifically bind n-3 FAs as agonist ligands. In this study, we examined the effects of n-3 FAs in human prostate cancer cell lines. Initial studies established that the long-chain n-3 FAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid, inhibit proliferation of DU145 cells in response to lysophosphatidic acid (LPA), a mitogenic lipid mediator. When added alone to serum-starved DU145 cells, EPA transiently activates signaling events, including p70S6K phosphorylation. However, when added 15 minutes prior to LPA, EPA suppresses LPA-induced activating phosphorylations of ERK, FAK, and p70S6K, and expression of the matricellular protein CCN1. The rapid onset of the inhibitory action of EPA suggested involvement of a GPCR. Further studies showed that DU145 and PC-3 cells express mRNA and protein for both FFA4 and FFA1. TUG-891 (4-[(4-fluoro-4′-methyl[1,1′-biphenyl]-2-yl)methoxy]-benzenepropanoic acid), a selective agonist for FFA4, exerts inhibitory effects on LPA- and epidermal growth factor–induced proliferation and migration, similar to EPA, in DU145 and PC-3 cells. The effects of TUG-891 and EPA are readily reversible. The FFA1/FFA4 agonist GW9508 (4-[[(3-phenoxyphenyl)methyl]amino]-benzenepropranoic acid) likewise inhibits proliferation at doses that block FFA4. Knockdown of FFA4 expression prevents EPA- and TUG-891–induced inhibition of growth and migration. Together, these results indicate that activation of FFA4 initiates signaling events that can inhibit growth factor–induced signaling, providing a novel mechanism for suppression of cancer cell proliferation. PMID:25491146

  20. A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis

    PubMed Central

    Lee, Jung Hoon; Wada, Taira; Febbraio, Maria; He, Jinhan; Matsubara, Tsutomu; Lee, Min Jae; Gonzalez, Frank J.; Xie, Wen

    2010-01-01

    Background & Aims The aryl hydrocarbon receptor (AhR) is a PAS domain transcription factor previously known as the “dioxin receptor” or “xenobiotic receptor.” The goal of this study is to determine the endobiotic role of AhR in hepatic steatosis. Methods Wild type, constitutively activated AhR (CA-AhR) transgenic, AhR null (AhR-/-), and fatty acid translocase CD36/FAT null (CD36-/-) mice were used to investigate the role of AhR in steatosis and the involvement of CD36 in the steatotic effect of AhR. The promoters of the mouse and human CD36 genes were cloned and their regulation by AhR was analyzed. Results Activation of AhR induced spontaneous hepatic steatosis characterized by the accumulation of triglycerides. The steatotic effect of AhR is likely due to the combined upregulation of CD36 and fatty acid transport proteins (FATPs), suppression of fatty acid oxidation, inhibition of hepatic export of triglycerides, increase in peripheral fat mobilization, and increased hepatic oxidative stress. Promoter analysis established CD36 as a novel transcriptional target of AhR. Activation of AhR in liver cells induced CD36 gene expression and enhanced fatty acid uptake. The steatotic effect of an AhR agonist was inhibited in CD36-/- mice. Conclusions Our study reveals a novel link between AhR-induced steatosis and the expression of CD36. Industrial or military exposures to dioxin and related compounds have been linked to increased prevalence of fatty liver in humans. Results from this study may help to establish AhR and its target CD36 as novel therapeutic and preventive targets for fatty liver disease. PMID:20303349

  1. Human sweet taste receptor mediates acid-induced sweetness of miraculin.

    PubMed

    Koizumi, Ayako; Tsuchiya, Asami; Nakajima, Ken-ichiro; Ito, Keisuke; Terada, Tohru; Shimizu-Ibuka, Akiko; Briand, Loïc; Asakura, Tomiko; Misaka, Takumi; Abe, Keiko

    2011-10-01

    Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity. PMID:21949380

  2. Human sweet taste receptor mediates acid-induced sweetness of miraculin

    PubMed Central

    Koizumi, Ayako; Tsuchiya, Asami; Nakajima, Ken-ichiro; Ito, Keisuke; Terada, Tohru; Shimizu-Ibuka, Akiko; Briand, Loïc; Asakura, Tomiko; Misaka, Takumi; Abe, Keiko

    2011-01-01

    Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity. PMID:21949380

  3. Excitatory amino acid receptors and synaptic transmission in the rat ventrobasal thalamus.

    PubMed

    Salt, T E

    1987-10-01

    1. Extracellular single-neurone recordings were made in the ventrobasal thalamus (v.b.t.) of urethane-anaesthetized rats with multi-barrel ionophoretic electrodes in order to test the hypothesis that excitatory amino acid receptors are involved in the responses of these neurones to stimulation of sensory afferents. 2. Responses of neurones to either physiological stimulation of hair and vibrissa follicle sensory afferents and to ionophoretically applied excitatory amino acids were challenged with the antagonists D-2-amino-5-phosphonovalerate (APV), kynurenate and gamma-D-glutamylaminomethyl sulphonate (GAMS). 3. In agreement with previous findings in other brain areas, ionophoretically applied APV was found to selectively antagonize responses of v.b.t. neurones to N-methylaspartate (NMA), whereas GAMS was found to be moderately kainate selective. Kynurenate was found to be relatively non-selective. 4. Responses of neurones to short-duration (10-20 ms) physiological stimulation of afferents were resistant to APV when this antagonist was applied with NMA-selective ionophoretic currents. In contrast, these APV currents were adequate to antagonize responses to maintained physiological stimulation. 5. The broad spectrum excitatory amino acid antagonist kynurenate was found to block synaptic responses of v.b.t. neurones to both short-duration and maintained stimuli when it was applied with currents which were sufficient to reduce responses to ionophoretic quisqualate. 6. GAMS was found to selectively block kainate responses in a proportion of the neurones tested. In such cases, there was little effect of the antagonist on the responses evoked by either short-duration or maintained sensory stimuli. 7. It is concluded that excitatory amino acid receptors of both the NMDA and non-NMDA type are involved in the synaptic responses of v.b.t. neurones to sensory afferent stimulation, and that the apparent synaptic pharmacology depends on the mode of stimulation of the afferent

  4. Effect of retinoic acid and ethanol on retinoic acid receptor beta and glial fibrillary acidic protein mRNA expression in human astrocytoma cells.

    PubMed

    Grummer, M A; Salih, Z N; Zachman, R D

    2000-11-17

    This work explores the hypothesis that perturbations caused by ethanol on the regulatory role of retinoids in brain development may be a mechanism involved in the neuropathology of fetal alcohol syndrome. The interaction of ethanol and retinoic acid (RA) on RA receptor (RAR) beta and glial fibrillary acidic protein (GFAP) mRNA expression is evaluated. In the U-373 MG astrocytoma, mRNA expression of RAR beta was increased and GFAP was decreased by RA. Ethanol decreased the expression of RAR beta mRNA, but increased that of GFAP. The RA-stimulated increase in RAR beta was not affected by the presence of ethanol. RA prevented the ethanol-induced increase in GFAP mRNA. Cycloheximide abolished only the GFAP response to ethanol. This work shows that an interrelationship between ethanol and RA exists in the astrocyte. PMID:11058790

  5. Pharmacodynamics of long-acting folic acid-receptor targeted ritonavir boosted atazanavir nanoformulations

    PubMed Central

    Puligujja, Pavan; Balkundi, Shantanu; Kendrick, Lindsey; Baldridge, Hannah; Hilaire, James; Bade, Aditya N.; Dash, Prasanta K.; Zhang, Gang; Poluektova, Larisa; Gorantla, Santhi; Liu, Xin-Ming; Ying, Tianlei; Feng, Yang; Wang, Yanping; Dimitrov, Dimiter S.; McMillan, JoEllyn M.; Gendelman, Howard E.

    2014-01-01

    Long-acting nanoformulated antiretroviral therapy (nanoART) that target monocyte-macrophage could improve the drug’s half-life and protein binding capacities while facilitating cell and tissue depots. To this end, ART nanoparticles that target the folic acid (FA) receptor and permit cell-based drug depots were examined using pharmacokinetic and pharmacodynamic (PD) tests. FA receptor-targeted poloxamer 407 nanocrystals, containing ritonavir-boosted atazanavir (ATV/r), significantly affected several therapeutic factors: drug bioavailability increased as much as 5 times and PD activity improved as much as 100 times. Drug particles administered to human peripheral blood lymphocyte reconstituted NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice and infected with HIV-1ADA at a tissue culture infective dose50 of 104 infectious viral particles/ml led to ATV/r drug concentrations that paralleled FA receptor beta staining in both the macrophage-rich parafollicular areas of spleen and lymph nodes. Drug levels were higher in these tissues than what could be achieved by either native drug or untargeted nanoART particles. The data also mirrored potent reductions in viral loads, tissue viral RNA and numbers of HIV-1p24+ cells in infected and treated animals. We conclude that FA-P407 coating of ART nanoparticles readily facilitate drug carriage and facilitate antiretroviral responses. PMID:25522973

  6. Cellular localization of retinoic acid receptor-gamma expression in normal and neoplastic skin.

    PubMed Central

    Finzi, E.; Blake, M. J.; Celano, P.; Skouge, J.; Diwan, R.

    1992-01-01

    Retinoids profoundly affect the normal growth and differentiation of epithelial tissues. Retinoic acid receptor-gamma (RAR-gamma) is a member of a family of retinoid receptors, and has been shown to be expressed almost exclusively in skin. However, little is known about the cellular localization of this receptor in human skin. The authors studied the expression of RAR-gamma in normal skin and human skin tumors by Northern blot analysis and in situ hybridization. RAR-gamma mRNA was detected in normal skin as well as in cultures of neonatal keratinocytes. Using an oligonucleotide specific for the RAR-gamma cDNA isoform 1 (RAR-gamma 1), RAR-gamma 1 mRNA was localized to all layers of the epidermis, the outer root sheath of hair follicles, follicular hair bulbs, eccrine and sebaceous glands. Basal cell carcinoma constitutively expressed gamma-1 mRNA and one of seven squamous cell carcinomas showed loss of gamma-1 mRNA expression, relative to adjacent epithelium. By contrast, normal melanocytic nevi and tumor-associated lymphocytes expressed little or no RAR-gamma mRNA. These results suggest that RAR-gamma 1 may play an important role in the maintenance and differentiation of normal epidermis and skin appendages. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1318641

  7. Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts.

    PubMed

    Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P

    2014-12-26

    Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca(2+) puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca(2+) puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts.

  8. Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts.

    PubMed

    Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P

    2014-12-26

    Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca(2+) puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca(2+) puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts. PMID:25542932

  9. The effect of folic acid on GABA(A)-B 1 receptor subunit.

    PubMed

    Vasquez, Kizzy; Kuizon, Salomon; Junaid, Mohammed; Idrissi, Abdeslem El

    2013-01-01

    Autism contains a spectrum of behavioral and cognitive disturbances of childhood development that is manifested by deficits in social interaction, impaired communication, repetitive behavior, and/or restricted interest. Much research has been dedicated to finding the genes that are responsible for autism, but less than 10% of the cases can be attributed to one gene. Autism prevalence has increased in the last decade and there may be environmental components that are leading to this increase. There are reports of disruption of epigenetic mechanisms controlling the regulation of gene expression as probable cause for autism. Folic acid (FA) is prescribed to women during pregnancy, and can cause epigenetic changes. GABAergic pathway is involved in inhibitory neurotransmission in the central nervous system and plays a crucial role during early embryonic development. Autism may entail defect or deregulation of the GABAergic receptor pathway in the brain. Gamma-aminobutyric acid (type A) beta 1 receptor (GABRB1) disruption has been implicated in autism. In the present study, we investigated GABRB1 expression in response to FA supplementation in neuronal cells. Western blot analysis showed GABRB1 protein levels increased in the FA-treated cells in a concentration-dependent manner. FA-dependent increased expression of GABRB1 was further confirmed at the mRNA level using quantitative RT-PCR. These results suggest that epigenetic control of gene expression may affect the expression of GABRB1 and disrupt inhibitory synaptic transmission during embryonic development. PMID:23392927

  10. Kynurenic acid, an aryl hydrocarbon receptor ligand, is elevated in serum of Zucker fatty rats

    PubMed Central

    Oxenkrug, G; Cornicelli, J; van der Hart, M; Roeser, J; Summergrad, P

    2016-01-01

    Obesity is an increasingly urgent global problem and the molecular mechanisms of obesity are not fully understood. Dysregulation of the tryptophan (Trp) – kynurenine (Kyn) metabolic pathway (TKP) have been suggested as a mechanism of obesity and described in obese humans and in animal models of obesity. However, to the best of our knowledge, TKP metabolism has not been studied in leptin-receptor-deficient Zucker fatty rats (ZFR) (fa/fa), the best-known and most widely used rat model of obesity. We were interested to determine if there are any deviations of TKP in ZFR. Concentrations of major TKP metabolites were evaluated (HPLC- MS method) in serum of ZFR (fa/fa) and age-matched lean rats (FA/-). Concentrations of kynurenic acid (KYNA) were 50% higher in ZFR than in lean rats (p<0.004, Mann-Whitney two-tailed test). Anthranilic acid (AA) concentrations, while elevated by 33%, did not reach statistical significance (p<0.04, one-tailed test). Elevated KYNA serum concentrations might contribute to development of obesity via KYNA-induced activation of aryl hydrocarbon receptor. Present results warrant further studies of KYNA and AA in ZFR and other animal models of obesity. PMID:27738521

  11. Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats

    SciTech Connect

    Yamada, Takanori; Obo, Yumi; Furukawa, Mami; Hotta, Mayuko; Yamasaki, Ayako; Honoki, Kanya; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2009-01-16

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 out of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP.

  12. Molecular mechanism of the assembly of an acid-sensing receptor ion channel complex.

    PubMed

    Yu, Yong; Ulbrich, Maximilian H; Li, Ming-Hui; Dobbins, Scott; Zhang, Wei K; Tong, Liang; Isacoff, Ehud Y; Yang, Jian

    2012-01-01

    Polycystic kidney disease (PKD) family proteins associate with transient receptor potential (TRP) channel family proteins to form functionally important complexes. PKD proteins differ from known ion channel-forming proteins and are generally thought to act as membrane receptors. Here we find that PKD1L3, a PKD protein, functions as a channel-forming subunit in an acid-sensing heteromeric complex formed by PKD1L3 and TRPP3, a TRP channel protein. Single amino-acid mutations in the putative pore region of both proteins alter the channel's ion selectivity. The PKD1L3/TRPP3 complex in the plasma membrane of live cells contains one PKD1L3 and three TRPP3. A TRPP3 C-terminal coiled-coil domain forms a trimer in solution and in crystal, and has a crucial role in the assembly and surface expression of the PKD1L3/TRPP3 complex. These results demonstrate that PKD subunits constitute a new class of channel-forming proteins, enriching our understanding of the function of PKD proteins and PKD/TRPP complexes. PMID:23212381

  13. Acid-responsive PEGylated doxorubicin prodrug nanoparticles for neuropilin-1 receptor-mediated targeted drug delivery.

    PubMed

    Song, Huijuan; Zhang, Ju; Wang, Weiwei; Huang, Pingsheng; Zhang, Yumin; Liu, Jianfeng; Li, Chen; Kong, Deling

    2015-12-01

    Self-assembled prodrug nanoparticles have demonstrated great promise in cancer chemotherapy. In the present study, we developed a new kind of prodrug nanoparticles for targeted drug delivery. PEGylated doxorubicin conjugate with an acid-cleavable cis-aconityl spacer was prepared. Then it was functionalized with a tumor-penetrating peptide, Cys-Arg-Gly-Asp-Lys (CRGDK), providing the prodrug nanoparticles with the specific binding ability to neurophilin-1 receptor. In acid mediums, doxorubicin could be released from the prodrug nanoparticles with an accumulative release around 60% through the acid-triggered hydrolysis of cis-aconityl bond and nanoparticle disassembly. Whereas, drug release was slow under a neutral pH and the accumulative drug release was less than 16%. In the cell culture tests, our prodrug nanoparticles showed enhanced endocytosis and cytotoxicity in cancer cells including HepG2, MCF-7 and MDA-MB-231 cells, but lower cytotoxicity in human cardiomyocyte H2C9. In the animal experiments, the prodrug nanoparticles were intravenously injected into Balb/c nude mice bearing MDA-MB-231 tumors. Enhanced drug penetration and accumulation in tumors, accompanying with a rapid early tumor-binding behavior, was observed after intravenous injection of the peptide modified prodrug nanoparticles. These data suggests that the acid-sensitive and tumor-targeting PEGylated doxorubicin prodrug nanoparticle may be an efficient drug delivery system for cancer chemotherapy.

  14. GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position.

    PubMed

    Kitamura, Hajime; Makide, Kumiko; Shuto, Akira; Ikubo, Masaya; Inoue, Asuka; Suzuki, Kensuke; Sato, Yusuke; Nakamura, Sho; Otani, Yuko; Ohwada, Tomohiko; Aoki, Junken

    2012-05-01

    GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position. PMID:22343749

  15. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias.

    PubMed

    Bolognini, Daniele; Moss, Catherine E; Nilsson, Karolina; Petersson, Annika U; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B; Milligan, Graeme

    2016-09-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11 Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  16. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias*

    PubMed Central

    Bolognini, Daniele; Moss, Catherine E.; Nilsson, Karolina; Petersson, Annika U.; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M.; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B.

    2016-01-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11. Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  17. Triphenyl phosphate-induced developmental toxicity in zebrafish: Potential role of the retinoic acid receptor

    PubMed Central

    Isales, Gregory M.; Hipszer, Rachel A.; Raftery, Tara D.; Chen, Albert; Stapleton, Heather M.; Volz, David C.

    2015-01-01

    Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) – a high-production volume organophosphate-based flame retardant – results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) – a nuclear receptor that regulates vertebrate heart morphogenesis – in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5-72 hours post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) – a primary TPP metabolite - were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) – a major target gene for RA-induced RAR activation in zebrafish – and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may interact with human RARs, we then exposed Chinese hamster ovary cells stably transfected with chimeric human RARα-, RARβ-, or RARγ to TPP in the presence of RA, and found that TPP significantly inhibited RA-induced luciferase activity in a concentration-dependent manner. Overall, our findings suggest that zebrafish RARs may be involved in mediating TPP-induced developmental toxicity, a mechanism of action that may have relevance to humans. PMID:25725299

  18. Effects of bile acids and the bile acid receptor FXR agonist on the respiratory rhythm in the in vitro brainstem medulla slice of neonatal Sprague-Dawley rats.

    PubMed

    Zhao, Cong; Wang, Xianbao; Cong, Yuling; Deng, Yi; Xu, Yijun; Chen, Aihua; Yin, Yanru

    2014-01-01

    Intrahepatic cholestasis of pregnancy is always accompanied by adverse fetal outcomes such as malfunctions of respiration. Farnesoid X receptor (FXR) plays a critical role in the homeostasis of bile acids. Thus, we are determined to explore the effects of farnesoid X receptor (FXR) and five bile acids on respiratory rhythm generation and modulation of neonatal rats. Spontaneous periodic respiratory-related rhythmical discharge activity (RRDA) was recorded from hypoglossal nerves during the perfusion of modified Krebs solution. Group 1-6 was each given GW4064 and five bile acids of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), cholic acid (CA) as well as ursodeoxycholic acid (UDCA) at different concentrations to identify their specific functions on respiratory rhythm modulations. Group 7 was applied to receive FXR blocker Z-guggulsterone and Z-guggulsterone with the above bile acids separately to explore the role of FXR in the respiratory rhythm modulation. Group 8 was given dimethyl sulfoxide (DMSO) as controls. Apart from UDCA, CDCA, DCA LCA and CA all exerted effects on RRDA recorded from hypoglossal nerves in a concentration-dependent manner. Respiratory cycle (RC), Inspiratory time (TI), Expiratory Time (TE) and Integral Amplitude (IA) were influenced and such effects could be reversed by Z-guggulsterone. FXR may contribute to the effects on the modulation of respiratory rhythm exerted by bile acids.

  19. Efficient Modulation of γ-Aminobutyric Acid Type A Receptors by Piperine Derivatives

    PubMed Central

    2014-01-01

    Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode voltage-clamp technique. GABAAR were expressed in Xenopus laevis oocytes. Structure–activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-induced chloride current modulation (IGABA-max = 1673% ± 146%, EC50 = 51.7 ± 9.5 μM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC50 = 13.8 ± 1.8 μM, IGABA-max = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABAAR modulators. PMID:24905252

  20. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    PubMed

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

  1. Tannic Acid Down-Regulates the Angiotensin Type 1 Receptor Through a MAPK-Dependent Mechanism

    PubMed Central

    Yesudas, Rekha; Gumaste, Upendra; Snyder, Russell

    2012-01-01

    In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5–100 μg/ml (7.34–58.78 μm) over a time period of 2–24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression. PMID:22322600

  2. Lysergic acid diethylamide (LSD) administration selectively downregulates serotonin2 receptors in rat brain.

    PubMed

    Buckholtz, N S; Zhou, D F; Freedman, D X; Potter, W Z

    1990-04-01

    A dosage regimen of lysergic acid diethylamide (LSD) that reliably produces behavioral tolerance in rats was evaluated for effects on neurotransmitter receptor binding in rat brain using a variety of radioligands selective for amine receptor subtypes. Daily administration of LSD [130 micrograms/kg (0.27 mumol/kg) intraperitoneally (IP)] for 5 days produced a decrease in serotonin2 (5-hydroxytryptamine2, 5-HT2) binding in cortex (measured 24 hours after the last drug administration) but did not affect binding to other receptor systems (5-HT1A, 5-HT1B, beta-adrenergic, alpha 1- or alpha 2-adrenergic, D2-dopaminergic) or to a recognition site for 5-HT uptake. The decrease was evident within 3 days of LSD administration but was not demonstrable after the first LSD dose. Following 5 days of LSD administration the decrease was still present 48 hours, but not 96 hours, after the last administration. The indole hallucinogen psilocybin [1.0 mg/kg (3.5 mumol/kg) for 8 days] also produced a significant decrease in 5HT2 binding, but neither the nonhallucinogenic analog bromo-LSD [1.3 mg/kg (2.4 mumol/kg) for 5 days] nor mescaline [10 mg/kg (40.3 mumol/kg) for 5 or 10 days] affected 5-HT2 binding. These observations suggest that LSD and other indole hallucinogens may act as 5-HT2 agonists at postsynaptic 5-HT2 receptors. Decreased 5-HT2 binding strikingly parallels the development and loss of behavioral tolerance seen with repeated LSD administration, but the decreased binding per se cannot explain the gamut of behavioral tolerance and cross-tolerance phenomena among the indole and phenylethylamine hallucinogens.

  3. Running increases neurogenesis without retinoic acid receptor activation in the adult mouse dentate gyrus.

    PubMed

    Aberg, Elin; Perlmann, Thomas; Olson, Lars; Brené, Stefan

    2008-01-01

    Both vitamin A deficiency and high doses of retinoids can result in learning and memory impairments, depression as well as decreases in cell proliferation, neurogenesis and cell survival. Physical activity enhances hippocampal neurogenesis and can also exert an antidepressant effect. Here we elucidate a putative link between running, retinoid signaling, and neurogenesis in hippocampus. Adult transgenic reporter mice designed to detect ligand-activated retinoic acid receptors (RAR) or retinoid X receptors (RXR) were used to localize the distribution of activated RAR or RXR at the single-cell level in the brain. Two months of voluntary wheel-running induced an increase in hippocampal neurogenesis as indicated by an almost two-fold increase in doublecortin-immunoreactive cells. Running activity was correlated with neurogenesis. Under basal conditions a distinct pattern of RAR-activated cells was detected in the granule cell layer of the dentate gyrus (DG), thalamus, and cerebral cortex layers 3-4 and to a lesser extent in hippocampal pyramidal cell layers CA1-CA3. Running did not change the number of RAR-activated cells in the DG. There was no correlation between running and RAR activation or between RAR activation and neurogenesis in the DG of hippocampus. Only a few scattered activated retinoid X receptors were found in the DG under basal conditions and after wheel-running, but RXR was detected in other areas such as in the hilus region of hippocampus and in layer VI of cortex cerebri. RAR agonists affect mood in humans and reduce neurogenesis, learning and memory in animal models. In our study, long-term running increased neurogenesis but did not alter RAR ligand activation in the DG in individually housed mice. Thus, our data suggest that the effects of exercise on neurogenesis and other plasticity changes in the hippocampal formation are mediated by mechanisms that do not involve retinoid receptor activation.

  4. γ-Aminbuturic Acid A Receptor Mitigates Homocysteine-Induced Endothelial Cell Permeability

    PubMed Central

    Tyagi, Neetu; Moshal, Karni S.; Tyagi, Suresh C.; Lominadze, David

    2010-01-01

    Many cerebrovascular disorders are accompanied by an increased homocysteine (Hcy) levels. We have previously shown that acute hyperhomocysteinemia (HHcy) leads to an increased microvascular permeability in the mouse brain. Hcy competitively binds to γ -aminbuturic acid (GABA) receptors and may increase vascular permeability by acting as an excitatory neurotransmitter. However, the role of GABA-A (GABAA) receptor in Hcy-induced endothelial cell (EC) permeability remains unclear. In the present study we attempted to determine the role of GABAA receptor and the possible mechanisms involved in Hcy-induced EC layer permeability. Mouse aortic and brain ECs were grown in Transwells and treated with 50 μM Hcy in the presence or absence of GABAA-specific agonist muscimol. Role of matrix metalloproteinase-9 (MMP-9) was determined using its activity inhibitor GM-6001. Involvement of extracellular signal-regulated kinase (ERK) signaling was assessed using its kinase activity inhibitors PD98059 or U0126. EC permeability to the known content of bovine serum albumin (BSA)-conjugated with Alexa Flour-488 was assessed by measuring fluorescence intensity of the solutes in the Transwell's lower chambers. It was found that Hcy induced the formation of filamentous actin (F-actin). Hcy-induced EC permeability to BSA was significantly decreased by GABA and muscimol treatments. Presence of MMP-9 or ERK kinase activity inhibitors restored the Hcy-induced EC permeability to its baseline level. The mediation BSA leakage through the ECs was further confirmed in the experiments where Hcy-induced alterations in transendothelial electrical resistance of confluent ECs were assessed. The data suggest that Hcy increases EC layer permeability through inhibition of GABAA receptor and F-actin formation, in part, by transducing ERK and MMP-9 activation. PMID:18080868

  5. Selective agonists of retinoic acid receptors: comparative toxicokinetics and embryonic exposure.

    PubMed

    Arafa, H M; Elmazar, M M; Hamada, F M; Reichert, U; Shroot, B; Nau, H

    2000-01-01

    Three biologically active synthetic retinoids were investigated that bind selectively to retinoic acid receptors RARs (alpha, beta and gamma). The retinoids were previously demonstrated to have different teratogenic effects in the mouse in terms of potency and regioselectivity. The teratogenic potency rank order (alpha >beta >gamma) was found to be more or less compatible with the receptor binding affinities and transactivation potencies of the retinoid ligands to their respective receptors. The RARalpha agonist (Am580; CD336) induced a wide spectrum of malformations; CD2019 (RARbeta agonist) and especially CD437 (RARgamma agonist) produced more restricted defects. In the current study we tried to address whether the differences in teratogenic effects are solely related to binding affinity and transactivation differences or also due to differences in embryonic exposure. Therefore, transplacental kinetics of the ligands were assessed following administration of a single oral dose of 15 mg/kg of either retinoid given to NMRI mice on day 11 of gestation. Am580 was rapidly transferred to the embryo resulting in the highest embryonic exposure [embryo to maternal plasma area under the time vs concentration curve (AUC)(0-24 h )ratio (E/M) was 1.7], in accordance with its highest teratogenic potency. The low placental transfer of CD2019 (E/M of 0.3) was compatible with its lower teratogenic potential. Of major interest was the finding that the CD437, though being least teratogenic, exhibited considerable embryonic exposure (E/M of 0.6). These findings suggest that both the embryonic exposure and receptor binding transactivation selectivity are crucial determinants of the teratogenicity of these retinoid ligands.

  6. Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

    PubMed Central

    Sung, Yun-Min; Wilkins, Angela D.; Rodriguez, Gustavo J.; Wensel, Theodore G.; Lichtarge, Olivier

    2016-01-01

    The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues. PMID:26979958

  7. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    SciTech Connect

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  8. Ligand-dependent coactivation of the human bile acid receptor FXR by the peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Savkur, Rajesh S; Thomas, Jeffrey S; Bramlett, Kelli S; Gao, Yunling; Michael, Laura F; Burris, Thomas P

    2005-01-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to play an important role in energy metabolism by coordinating transcriptional programs involved in mitochondrial biogenesis, adaptive thermogenesis, gluconeogenesis, and fatty acid oxidation. PGC-1alpha also plays a crucial role in cholesterol metabolism by serving as a coactivator of the liver X receptor-alpha and inducing the expression of cholesterol 7-alpha-hydroxylase. Here, we demonstrate that PGC-1alpha also functions as an effective coactivator of farnesoid X receptor (FXR), the bile acid receptor. Transient cotransfection assays demonstrate that PGC-1alpha enhances ligand-mediated FXR transcription when either full-length FXR or Gal4 DNA binding domain-FXR-ligand binding domain chimeras were analyzed. Mammalian two-hybrid analyses, glutathione S-transferase affinity chromatography and biochemical coactivator recruitment assays demonstrate ligand-dependent interaction between the two proteins both in vivo and in vitro. PGC-1alpha-mediated coactivation of FXR was highly ligand-dependent and absolutely required an intact activation function-2 (AF-2) domain of FXR and the LXXLL motif in PGC-1alpha. The integrity of the charge clamp was required, further illustrating the role of the ligand binding domain of FXR in PGC-1alpha recognition. Together, these results indicate that PGC-1alpha functions as a potent coactivator for FXR and further implicates its role in the regulation of genes that are involved in bile acid and lipid metabolism.

  9. Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors.

    PubMed

    Liu, F; Wan, Q; Pristupa, Z B; Yu, X M; Wang, Y T; Niznik, H B

    2000-01-20

    GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.

  10. [The underwater EOG of the frog Rana temporaria to stimulation of the olfactory receptors with solutions of fragrances and amino acids].

    PubMed

    Kruzhalov, N B

    1991-01-01

    Studies have been made on the effectiveness of 16 amino acids and 9 odorants in olfactory perception of the frog Rana temporaria. It was shown that the effectiveness of amino acids is close to that of odorants. Basic amino acids and cysteine were more effective than other amino acids. Sensitivity of the olfactory receptors to amino acids in frogs is lower than in fishes.

  11. Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome.

    PubMed

    Meslin, Camille; Desert, Colette; Callebaut, Isabelle; Djari, Anis; Klopp, Christophe; Pitel, Frédérique; Leroux, Sophie; Martin, Pascal; Froment, Pascal; Guilbert, Edith; Gondret, Florence; Lagarrigue, Sandrine; Monget, Philippe

    2015-04-24

    Free fatty acid receptors (FFAR) belong to a family of five G-protein coupled receptors that are involved in the regulation of lipid metabolism, so that their loss of function increases the risk of obesity. The aim of this study was to determine the expansion of genes encoding paralogs of FFAR2 in the chicken, considered as a model organism for developmental biology and biomedical research. By estimating the gene copy number using quantitative polymerase chain reaction, genomic DNA resequencing, and RNA sequencing data, we showed the existence of 23 ± 1.5 genes encoding FFAR2 paralogs in the chicken genome. The FFAR2 paralogs shared an identity from 87.2% up to 99%. Extensive gene conversion was responsible for this high degree of sequence similarities between these genes, and this concerned especially the four amino acids known to be critical for ligand binding. Moreover, elevated nonsynonymous/synonymous substitution ratios on some amino acids within or in close-vicinity of the ligand-binding groove suggest that positive selection may have reduced the effective rate of gene conversion in this region, thus contributing to diversify the function of some FFAR2 paralogs. All the FFAR2 paralogs were located on a microchromosome in a same linkage group. FFAR2 genes were expressed in different tissues and cells such as spleen, peripheral blood mononuclear cells, abdominal adipose tissue, intestine, and lung, with the highest rate of expression in testis. Further investigations are needed to determine whether these chicken-specific events along evolution are the consequence of domestication and may play a role in regulating lipid metabolism in this species.

  12. Effect of dexamethasone and oxygen exposure on neonatal rat lung retinoic acid receptor proteins.

    PubMed

    McMenamy, K R; Anderson, M J; Zachman, R D

    1994-10-01

    Retinol deficiency in animal models results in histopathologic airway changes that appear similar to those found in human premature infants with bronchopulmonary dysplasia (BPD). Dexamethasone (DEX), a steroid now often used in the treatment of BPD, might potentially affect lung vitamin A homeostasis since it alters serum and liver retinoid stores in certain models. Our objective was to determine the effect of DEX on neonatal rat lung retinoid status and the binding of retinoic acid (RA) to cytosolic and nuclear receptor proteins. We examined this effect both in room air and when the animals breathed 95% oxygen (O2). Twenty-four 1-day-old rat pups received either 1 microgram/g DEX subcutaneously, an equal volume of normal saline (NS) subcutaneously at 0 (start experiment time), 24, and 48 hours, or no injection at all, and were sacrificed at 72 hours. Twelve rats in each treatment group were housed in room air and 12 in each group were exposed to > 95% O2 for the 3 day period. Lung and liver were analyzed for retinyl palmitate (RP). Nuclear retinoic acid receptor (RAR) and cellular retinoic acid binding protein (CRABP) were measured by specific binding assays. DEX decreased liver RP by 33-55% and rat pup lung RP by over 60%; it also decreased lung RAR binding (mean dpm/microgram protein +/- SEM) in both room air and oxygen groups: Air (11.2 +/- 1.0) vs. Air/DEX (4.6 +/- 1.3, n = 6; P < 0.01), and O2 (18.2 +/- 0.6) vs. O2/DEX (3.2 +/- 0.6, n = 6; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7838622

  13. Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity.

    PubMed

    Yu, Jinghua; Lo, Jane-L; Huang, Li; Zhao, Annie; Metzger, Edward; Adams, Alan; Meinke, Peter T; Wright, Samuel D; Cui, Jisong

    2002-08-30

    Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.

  14. (+)Lysergic acid diethylamide, but not its nonhallucinogenic congeners, is a potent serotonin 5HT1C receptor agonist

    SciTech Connect

    Burris, K.D.; Breeding, M.; Sanders-Bush, E. )

    1991-09-01

    Activation of central serotonin 5HT2 receptors is believed to be the primary mechanism whereby lysergic acid diethylamide (LSD) and other hallucinogens induce psychoactive effects. This hypothesis is based on extensive radioligand binding and electrophysiological and behavioral studies in laboratory animals. However, the pharmacological profiles of 5HT2 and 5HT1C receptors are similar, making it difficult to distinguish between effects due to activation of one or the other receptor. For this reason, it was of interest to investigate the interaction of LSD with 5HT1C receptors. Agonist-stimulated phosphoinositide hydrolysis in rat choroid plexus was used as a direct measure of 5HT1C receptor activation. (+)LSD potently stimulated phosphoinositide hydrolysis in intact choroid plexus and in cultures of choroid plexus epithelial cells, with EC50 values of 9 and 26 nM, respectively. The effect of (+)LSD in both systems was blocked by 5HT receptor antagonists with an order of activity consistent with interaction at 5HT1C receptors. Neither (+)-2-bromo-LSD nor lisuride, two nonhallucinogenic congeners of LSD, were able to stimulate 5HT1C receptors in cultured cells or intact choroid plexus. In contrast, lisuride, like (+)LSD, is a partial agonist at 5HT2 receptors in cerebral cortex slices and in NIH 3T3 cells transfected with 5HT2 receptor cDNA. The present finding that (+)LSD, but not its nonhallucinogenic congeners, is a 5HT1C receptor agonist suggests a possible role for these receptors in mediating the psychoactive effects of LSD.

  15. Agonist-directed signaling of serotonin 5-HT2C receptors: differences between serotonin and lysergic acid diethylamide (LSD).

    PubMed

    Backstrom, J R; Chang, M S; Chu, H; Niswender, C M; Sanders-Bush, E

    1999-08-01

    For more than 40 years the hallucinogen lysergic acid diethylamide (LSD) has been known to modify serotonin neurotransmission. With the advent of molecular and cellular techniques, we are beginning to understand the complexity of LSD's actions at the serotonin 5-HT2 family of receptors. Here, we discuss evidence that signaling of LSD at 5-HT2C receptors differs from the endogenous agonist serotonin. In addition, RNA editing of the 5-HT2C receptor dramatically alters the ability of LSD to stimulate phosphatidylinositol signaling. These findings provide a unique opportunity to understand the mechanism(s) of partial agonism.

  16. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste. PMID:26701297

  17. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

  18. Analogues of gamma-aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) substituted in the 2 position as GABAC receptor antagonists.

    PubMed

    Chebib, M; Vandenberg, R J; Johnston, G A

    1997-12-01

    1. gamma-Aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) have been shown to activate GABAC receptors. In this study, a range of C2, C3, C4 and N-substituted GABA and TACA analogues were examined for activity at GABAC receptors. 2. The effects of these compounds were examined by use of electrophysiological recording from Xenopus oocytes expressing the human rho 1 subunit of GABAC receptors with the two-electrode voltage-clamp method. 3. trans-4-Amino-2-fluorobut-2-enoic acid was found to be a potent agonist (KD = 2.43 microM). In contrast, trans-4-amino-2-methylbut-2-enoic acid was found to be a moderately potent antagonist (IC50 = 31.0 microM and KB = 45.5 microM). These observations highlight the possibility that subtle structural substitutions may change an agonist into an antagonist. 4. 4-Amino-2-methylbutanoic acid (KD = 189 microM), 4-amino-2-methylenebutanoic acid (KD = 182 microM) and 4-amino-2-chlorobutanoic acid (KD = 285 microM) were weak partial agonists. The intrinsic activities of these compounds were 12.1%, 4.4% and 5.2% of the maximal response of GABA, respectively. These compounds more effectively blocked the effects of the agonist, GABA, giving rise to KB values of 53 microM and 101 microM, respectively. 5. The sulphinic acid analogue of GABA, homohypotaurine, was found to be a potent partial agonist (KD = 4.59 microM, intrinsic activity 69%). 6. It was concluded that substitution of a methyl or a halo group in the C2 position of GABA or TACA is tolerated at GABAC receptors. However, there was dramatic loss of activity when these groups were substituted at the C3, C4 and nitrogen positions of GABA and TACA. 7. Molecular modelling studies on a range of active and inactive compounds indicated that the agonist/competitive antagonist binding site of the GABAC receptor may be smaller than that of the GABAA and GABAB receptors. It is suggested that only compounds that can attain relatively flat conformations may bind to the GABAC receptor

  19. International Union of Basic and Clinical Pharmacology. LXXXII: Nomenclature and Classification of Hydroxy-carboxylic Acid Receptors (GPR81, GPR109A, and GPR109B).

    PubMed

    Offermanns, Stefan; Colletti, Steven L; Lovenberg, Timothy W; Semple, Graeme; Wise, Alan; IJzerman, Adriaan P

    2011-06-01

    The G-protein-coupled receptors GPR81, GPR109A, and GPR109B share significant sequence homology and form a small group of receptors, each of which is encoded by clustered genes. In recent years, endogenous ligands for all three receptors have been described. These endogenous ligands have in common that they are hydroxy-carboxylic acid metabolites, and we therefore have proposed that this receptor family be named hydroxy-carboxylic acid (HCA) receptors. The HCA(1) receptor (GPR81) is activated by 2-hydroxy-propanoic acid (lactate), the HCA(2) receptor (GPR109A) is a receptor for the ketone body 3-hydroxy-butyric acid, and the HCA(3) receptor (GPR109B) is activated by the β-oxidation intermediate 3-hydroxy-octanoic acid. HCA(1) and HCA(2) receptors are found in most mammalian species, whereas the HCA(3) receptor is present only in higher primates. The three receptors have in common that they are expressed in adipocytes and are coupled to G(i)-type G-proteins mediating antilipolytic effects in fat cells. HCA(2) and HCA(3) receptors are also expressed in a variety of immune cells. HCA(2) is a receptor for the antidyslipidemic drug nicotinic acid (niacin) and related compounds, and there is an increasing number of synthetic ligands mainly targeted at HCA(2) and HCA(3) receptors. The aim of this article is to give an overview on the discovery and pharmacological characterization of HCAs, and to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature. We will also discuss open questions regarding this receptor family as well as their physiological role and therapeutic potential.

  20. Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine.

    PubMed

    Kim, Insook; Ahn, Sung-Hoon; Inagaki, Takeshi; Choi, Mihwa; Ito, Shinji; Guo, Grace L; Kliewer, Steven A; Gonzalez, Frank J

    2007-12-01

    Bile acid concentrations are controlled by a feedback regulatory pathway whereby activation of the farnesoid X receptor (FXR) represses transcription of both the CYP7A1 gene, encoding the rate-limiting enzyme in the classic bile acid synthesis pathway, and the CYP8B1 gene, required for synthesis of cholic acid. The tissue-specific roles of FXR were examined using liver- and intestine-specific FXR-null models. FXR deficiency in either liver (Fxr DeltaL) or intestine (Fxr DeltaIE) increased bile acid pool size. Treatment with the FXR-selective agonist GW4064 significantly repressed CYP7A1 in Fxr DeltaL mice but not Fxr DeltaIE mice, demonstrating that activation of FXR in intestine but not liver is required for short-term repression of CYP7A1 in liver. This intestinal-specific effect of FXR is likely mediated through induction of the hormone FGF15, which suppresses CYP7A1. In comparison to CYP7A1, FXR-mediated repression of CYP8B1 was more dependent on the presence of FXR in liver and less dependent on its presence in intestine. Consistent with these findings, recombinant FGF15 repressed CYP7A1 mRNA levels without affecting CYP8B1 expression. These data provide evidence that FXR-mediated repression of bile acid synthesis requires the complementary actions of FXR in both liver and intestine and reveal mechanistic differences in feedback repression of CYP7A1 and CYP8B1.

  1. Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs.

    PubMed

    Zhang, Xu-Fu; Dai, Ying-Chun; Zhong, Weiming; Tan, Ming; Lv, Zhi-Ping; Zhou, Ying-Chun; Jiang, Xi

    2012-02-15

    Noroviruses (NoVs) are the leading cause of viral acute gastroenteritis affecting people of all ages worldwide. The disease is difficult to control due to its widespread nature and lack of an antiviral or vaccine. NoV infection relies on the interaction of the viruses with histo-blood group antigens (HBGAs) as host receptors. Here we investigated inhibition effects of Chinese medicinal herbs against NoVs binding to HBGAs for potential antivirals against NoVs. Blocking assays was performed using the NoV protrusion (P) protein as NoV surrogate and saliva as HBGAs. Among 50 clinically effective Chinese medicinal herbs against gastroenteritis diseases, two herbs were found highly effective. Chinese Gall blocked NoV P dimer binding to type A saliva at IC(50)=5.35 μg/ml and to B saliva at IC(50)=21.7 μg/ml. Similarly, Pomegranate blocked binding of NoV P dimer to type A saliva at IC(50)=15.59 μg/ml and B saliva at IC(50)=66.67 μg/ml. Literature data on preliminary biochemistry analysis showed that tannic acid is a common composition in the extracts of the two herbs, so we speculate that it might be the effective compound and further studies using commercially available, highly purified tannic acid confirmed the tannic acid as a strong inhibitor in the binding of NoV P protein to both A and B saliva (IC(50)≈0.1 μM). In addition, we tested different forms of hydrolysable tannins with different alkyl esters, including gallic acid, ethyl gallate, lauryl gallate, octyl gallate and propyl gallate. However, none of these tannins-derivatives revealed detectable inhibiting activities. Our data suggested that tannic acid is a promising candidate antiviral against NoVs.

  2. Chlorogenic Acid Improves Late Diabetes through Adiponectin Receptor Signaling Pathways in db/db Mice

    PubMed Central

    Jin, Shasha; Chang, Cuiqing; Zhang, Lantao; Liu, Yang; Huang, Xianren; Chen, Zhimin

    2015-01-01

    The aim of this study was to examine the effects of chlorogenic acid (CGA) on glucose and lipid metabolism in late diabetic db/db mice, as well as on adiponectin receptors and their signaling molecules, to provide evidence for CGA in the prevention of type 2 diabetes. We randomly divided 16 female db/db mice into db/db-CGA and db/db-control (CON) groups equally; db/m mice were used as control mice. The mice in both the db/db-CGA and db/m-CGA groups were administered 80 mg/kg/d CGA by lavage for 12 weeks, whereas the mice in both CON groups were given equal volumes of phosphate-buffered saline (PBS) by lavage. At the end of the intervention, we assessed body fat and the parameters of glucose and lipid metabolism in the plasma, liver and skeletal muscle tissues as well as the levels of aldose reductase (AR) and transforming growth factor-β1 (TGF-β1) in the kidneys and measured adiponectin receptors and the protein expression of their signaling molecules in liver and muscle tissues. After 12 weeks of intervention, compared with the db/db-CON group, the percentage of body fat, fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) in the db/db-CGA group were all significantly decreased; TGF-β1 protein expression and AR activity in the kidney were both decreased; and the adiponectin level in visceral adipose was increased. The protein expression of adiponectin receptors (ADPNRs), the phosphorylation of AMP-activated protein kinase (AMPK) in the liver and muscle, and the mRNA and protein levels of peroxisome proliferator-activated receptor alpha (PPAR-α) in the liver were all significantly greater. CGA could lower the levels of fasting plasma glucose and HbA1c during late diabetes and improve kidney fibrosis to some extent through the modulation of adiponectin receptor signaling pathways in db/db mice. PMID:25849026

  3. Expression of retinoic acid receptor alpha mRNA in human leukemia cells.

    PubMed

    Largman, C; Detmer, K; Corral, J C; Hack, F M; Lawrence, H J

    1989-07-01

    The expression of the newly described human retinoic acid receptor alpha (RAR alpha) in six nonlymphoid and six lymphoid leukemia cell lines and nine freshly obtained samples of leukemia cells from patients with acute nonlymphoid leukemia was assessed by Northern blot analysis, using a full length cDNA clone of RAR alpha as probe. RAR alpha was expressed in all 12 cell lines and in all fresh leukemia samples as two major transcripts of 2.6 and 3.5 kb in size. Levels of RAR alpha expression and transcript sizes in retinoid-sensitive cells (such as HL60 or fresh promyelocytic leukemia cells) were not different from those in other samples. Moreover, expression of RAR alpha was not significantly modulated by exposure to cis-retinoic acid (cisRA) in either cisRA-responsive or unresponsive cells. By using a 3' fragment of the RAR alpha gene as a probe, we confirmed that the transcripts visualized did not represent the homologous RAR beta gene. RAR alpha appears to be expressed in most human leukemia cells regardless of the type of biologic response to retinoic acid.

  4. Effects of Angiotensin II Receptor Blockers on Metabolism of Arachidonic Acid via CYP2C8.

    PubMed

    Senda, Asuna; Mukai, Yuji; Toda, Takaki; Hayakawa, Toru; Yamashita, Miki; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2015-01-01

    Arachidonic acid (AA) is metabolized to epoxyeicosatrienoic acids (EETs) via cytochrome enzymes such as CYP 2C9, 2C8 and 2J2. EETs play a role in cardioprotection and regulation of blood pressure. Recently, adverse reactions such as sudden heart attack and fatal myocardial infarction were reported among patients taking angiotensin II receptor blockers (ARBs). As some ARBs have affinity for these CYP enzymes, metabolic inhibition of AA by ARBs is a possible cause for the increase in cardiovascular events. In this study, we quantitatively investigated the inhibitory effects of ARBs on the formation of EETs and further metabolites, dihydroxyeicosatrienoic acids (DHETs), from AA via CYP2C8. In incubations with recombinant CYP2C8 in vitro, the inhibitory effects were compared by measuring EETs and DHETs by HPLC-MS/MS. Inhibition of AA metabolism by ARBs was detected in a concentration-dependent manner with IC50 values of losartan (42.7 µM), telmisartan (49.5 µM), irbesartan (55.6 µM), olmesartan (66.2 µM), candesartan (108 µM), and valsartan (279 µM). Losartan, telmisartan and irbesartan, which reportedly accumulate in the liver and kidneys, have stronger inhibitory effects than other ARBs. The lower concentration of EETs leads to less protective action on the cardiovascular system and a higher incidence of adverse effects such as sudden heart attack and myocardial infarction in patients taking ARBs. PMID:26632190

  5. Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient mice

    PubMed Central

    Chung, Sanny S W; Choi, Cindy; Wang, Xiangyuan; Hallock, Loretta; Wolgemuth, Debra J

    2009-01-01

    Retinoic acid receptor alpha (RARα)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In the present study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell-cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RARα-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RARα-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RARα-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a down-regulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. PMID:19937743

  6. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors

    PubMed Central

    Pal Choudhuri, Shreoshi; Delay, Rona J.; Delay, Eugene R.

    2015-01-01

    Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5’ ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5’ monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex. PMID:26110622

  7. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

    PubMed

    Pal Choudhuri, Shreoshi; Delay, Rona J; Delay, Eugene R

    2015-01-01

    Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  8. The role of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

    PubMed

    Sawamura, Ryo; Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

    2015-03-01

    Clarification of the mechanism of chickens' taste sense will provide meaningful information for creating and improving new feedstuff for chickens, because the character of taste receptors in oral tissues affects feeding behavior in animals. Although fatty acids are partly recognized via G-protein coupled receptor 120 (GPR120) for fat taste in mammalian oral tissues, the fat taste receptor of chickens has not been elucidated. Here we cloned chicken GPR120 (cGPR120) from the chicken palate, which contains taste buds. By using Ca(2+) imaging methods, we identified oleic acid and linoleic acid as cGPR120 agonists. Interestingly, in a behavioral study the chickens preferred corn oil-rich feed over mineral oil (control oil)-rich feed. Because corn oil contains high amounts of oleic acid and linoleic acid, this result was thought to be reasonable. Taken together, the present results suggest that cGPR120 is one of the functional fat taste receptors in chickens.

  9. Omega-3 fatty acids are able to modulate the painful symptoms associated to cyclophosphamide-induced-hemorrhagic cystitis in mice.

    PubMed

    Freitas, Raquel D S; Costa, Kesiane M; Nicoletti, Natália F; Kist, Luiza W; Bogo, Maurício R; Campos, Maria M

    2016-01-01

    This study investigated the effects of the long-term dietary fish oil supplementation or the acute administration of the omega-3 fatty acid docosahexaenoic acid (DHA) in the mouse hemorrhagic cystitis (HC) induced by the anticancer drug cyclophosphamide (CYP). HC was induced in mice by a single CYP injection (300mg/kg ip). Animals received four different diets containing 10% and 20% of corn or fish oil, during 21days. Separated groups received DHA by ip (1μmol/kg) or intrathecal (i.t.; 10μg/site) routes, 1h or 15min before CYP. The behavioral tests (spontaneous nociception and mechanical allodynia) were carried out from 1h to 6h following CYP injection. Bladder inflammatory changes, blood cell counts and serum cytokines were evaluated after euthanasia (at 6h). Immunohistochemistry analysis was performed for assessing spinal astrocyte and microglia activation or GPR40/FFAR1 expression. Either fish oil supplementation or DHA treatment (ip and i.t.) markedly prevented visceral pain, without affecting CYP-evoked bladder inflammatory changes. Moreover, systemic DHA significantly prevented the neutrophilia/lymphopenia caused by CYP, whereas this fatty acid did not significantly affect serum cytokines. DHA also modulated the spinal astrocyte activation and the GPR40/FFAR1 expression. The supplementation with fish oil enriched in omega-3 fatty acids or parenteral DHA might be interesting nutritional approaches for cancer patients under chemotherapy schemes with CYP.

  10. Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

    PubMed Central

    Casanova, J; Helmer, E; Selmi-Ruby, S; Qi, J S; Au-Fliegner, M; Desai-Yajnik, V; Koudinova, N; Yarm, F; Raaka, B M; Samuels, H H

    1994-01-01

    The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor

  11. Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity.

    PubMed Central

    Durand, B; Saunders, M; Gaudon, C; Roy, B; Losson, R; Chambon, P

    1994-01-01

    A motif essential for the transcriptional activation function 2 (AF-2) present in the E region of retinoic acid receptor (RAR) alpha and 9-cis retinoic acid receptor (RXR) alpha has been characterized as an amphipathic alpha-helix whose main features are conserved between transcriptionally active members of the nuclear receptor superfamily. This conserved motif, which can activate autonomously in the absence of ligand in animal and yeast cells, can be swapped between nuclear receptors without affecting the ligand dependency for activation of transcription, thus indicating that a ligand-dependent conformational change is necessary to reveal the AF-2 activation potential within the E region of the nuclear receptor. Interestingly, we show that the precise nature of the direct repeat response element to which RAR/RXR heterodimers are bound can affect the activity of the AF-2s of the heterodimeric partners, as well as the relative efficiency with which all-trans and 9-cis retinoic acids activate the RAR partner. Images PMID:7957103

  12. Gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study

    SciTech Connect

    Shin, C.; Pedersen, H.B.; McNamara, J.O.

    1985-10-01

    Quantitative radiohistochemistry was utilized to study alterations of gamma-aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were (TH) muscimol and (TH)flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hr but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. The authors suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals.

  13. Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.

    PubMed

    Benacquista, B L; Sharma, M R; Samsó, M; Zorzato, F; Treves, S; Wagenknecht, T

    2000-03-01

    We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.

  14. Identification of sulfated oligosialic acid units in the O-linked glycan of the sea urchin egg receptor for sperm.

    PubMed

    Kitazume-Kawaguchi, S; Inoue, S; Inoue, Y; Lennarz, W J

    1997-04-15

    The Strongylocentrotus purpuratus sea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfated O-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that both N- and O-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of the N-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to the O-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin, Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO(4)-)-9Neu5Gc alpha2(-->5-O(glycolyl)Neu5Gc alpha2-->)n. Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that is O-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from the S. purpuratus egg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.

  15. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    PubMed Central

    Hudson, Brian D.; Christiansen, Elisabeth; Tikhonova, Irina G.; Grundmann, Manuel; Kostenis, Evi; Adams, David R.; Ulven, Trond; Milligan, Graeme

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC50 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.—Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs. PMID:22919070

  16. Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro.

    PubMed

    Zhang, Qiao; Wang, Shifeng; Yu, Yangyang; Sun, Shengnan; Zhang, Yuxin; Zhang, Yanling; Yang, Wei; Li, Shiyou; Qiao, Yanjiang

    2016-01-01

    Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR. PMID:27490540

  17. Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro

    PubMed Central

    Zhang, Qiao; Wang, Shifeng; Yu, Yangyang; Sun, Shengnan; Zhang, Yuxin; Zhang, Yanling; Yang, Wei; Li, Shiyou; Qiao, Yanjiang

    2016-01-01

    Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR. PMID:27490540

  18. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent

    2016-01-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  19. Loss of lysophosphatidic acid receptor-3 enhances cell migration in rat lung tumor cells

    SciTech Connect

    Hayashi, Mai; Okabe, Kyoko; Yamawaki, Yasuna; Teranishi, Miki; Honoki, Kanya; Mori, Toshio; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2011-02-18

    Research highlights: {yields} Loss of the Lpar3 expression due to aberrant DNA methylation occurred in rat lung tumor cells. {yields} The Lpar3 inhibited cell migration of rat lung tumor cells. {yields} The Lpar3 may act as a negative regulator of rat lung tumor cells. -- Abstract: Lysophosphatidic acid (LPA) indicates several biological effects, such as cell proliferation, differentiation and migration. LPA interacts with G protein-coupled transmembrane LPA receptors. In our previous report, we detected that loss of the LPA receptor-1 (Lpar1) expression is due to its aberrant DNA methylation in rat tumor cell lines. In this study, to assess an involvement of the other LPA receptor, Lpar3, in the pathogenesis of rat lung tumor cells, we measured the expression levels of the Lpar3 gene and its DNA methylation status by reverse transcription (RT)-polymerase chain reaction (PCR) and bisulfite sequencing analyses, respectively. RLCNR lung adenocarcinoma cells showed reduced expression of the Lpar3, compared with normal lung tissues. In the 5' upstream region of the Lpar3, normal lung tissues were unmethylated. By contrast, RLCNR cells were highly methylated, correlating with reduced expressions of the Lpar3. Based on these results, we generated the Lpar3-expressing RLCNR-a3 cells and measured the cell migration ability. Interestingly, the cell migration of RLCNR-a3 cells was significantly lower than that of RLCNR cells. This study suggests that loss of the Lpar3 due to aberrant DNA methylation may be involved in the progression of rat lung tumor cells.

  20. Sphingosine kinase 1 is upregulated with lysophosphatidic acid receptor 2 in human colorectal cancer

    PubMed Central

    Shida, Dai; Inoue, Satoru; Yoshida, Yuki; Kodaka, Atsushi; Tsuji, Tsutomu; Tsuiji, Makoto

    2016-01-01

    AIM: To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer. METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2. RESULTS: Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson’s correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage. CONCLUSION: Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer. PMID:26937138

  1. Lysophosphatidic acid induces anxiety-like behavior via its receptors in mice.

    PubMed

    Yamada, Misa; Tsukagoshi, Mai; Hashimoto, Tomio; Oka, Jun-Ichiro; Saitoh, Akiyoshi; Yamada, Mitsuhiko

    2015-03-01

    Lysophosphatidic acid (LPA) is a potent bioactive lipid mediator with diverse biological properties. We previously found altered expression of the LPA-related genes in rodents after treatment with sertraline, which is widely used to treat anxiety disorders and depression. However, little is known about the behavioral effects of LPA. In the present study, we investigated the behavioral effects of intracerebroventricular injection of LPA in adult mice. LPA did not significantly affect spontaneous locomotor activity, suggesting that LPA does not induce hyperactivity, ataxia, or sedation. We next investigated the emotional effects of LPA via the hole-board test. LPA significantly increased the number of head-dips in a dose- and time-related manner. A significant induction of head-dip counts occurred 15 and 30 min after LPA administration. To clarify the involvement of LPA receptors, we examined the effect of the non-selective LPA1-4 receptor antagonist, 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA) co-administered with LPA. BrP-LPA dose-dependently inhibited LPA-induced head-dip counts. We next investigated anxiety-like behavior via the elevated plus-maze test. LPA significantly reduced the percentage of time spent in the open arms and BrP-LPA dose-dependently inhibited this anxiety-like behavior. In conclusion, LPA induced anxiety-like behavior in mice via LPA receptors. Our results suggest that LPA signaling plays an important role in regulating anxiety in mice.

  2. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    SciTech Connect

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki; Kawada, Teruo

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  3. Improvement in Aqueous Solubility of Retinoic Acid Receptor (RAR) Agonists by Bending the Molecular Structure.

    PubMed

    Hiramatsu, Michiaki; Ichikawa, Yuki; Tomoshige, Shusuke; Makishima, Makoto; Muranaka, Atsuya; Uchiyama, Masanobu; Yamaguchi, Takao; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-08-01

    Aqueous solubility is a key requirement for many functional molecules, e. g., drug candidates. Decrease of the partition coefficient (log P) by chemical modification, i.e., introduction of hydrophilic group(s) into molecules, is a classical strategy for improving aqueous solubility. We have been investigating alternative strategies for improving the aqueous solubility of pharmaceutical compounds by disrupting intermolecular interactions. Here, we show that introducing a bend into the molecular structure of retinoic acid receptor (RAR) agonists by changing the substitution pattern from para to meta or ortho dramatically enhances aqueous solubility by up to 890-fold. We found that meta analogs exhibit similar hydrophobicity to the parent para compound, and have lower melting points, supporting the idea that the increase of aqueous solubility was due to decreased intermolecular interactions in the solid state as a result of the structural changes.

  4. Improvement in Aqueous Solubility of Retinoic Acid Receptor (RAR) Agonists by Bending the Molecular Structure.

    PubMed

    Hiramatsu, Michiaki; Ichikawa, Yuki; Tomoshige, Shusuke; Makishima, Makoto; Muranaka, Atsuya; Uchiyama, Masanobu; Yamaguchi, Takao; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-08-01

    Aqueous solubility is a key requirement for many functional molecules, e. g., drug candidates. Decrease of the partition coefficient (log P) by chemical modification, i.e., introduction of hydrophilic group(s) into molecules, is a classical strategy for improving aqueous solubility. We have been investigating alternative strategies for improving the aqueous solubility of pharmaceutical compounds by disrupting intermolecular interactions. Here, we show that introducing a bend into the molecular structure of retinoic acid receptor (RAR) agonists by changing the substitution pattern from para to meta or ortho dramatically enhances aqueous solubility by up to 890-fold. We found that meta analogs exhibit similar hydrophobicity to the parent para compound, and have lower melting points, supporting the idea that the increase of aqueous solubility was due to decreased intermolecular interactions in the solid state as a result of the structural changes. PMID:27378357

  5. Dynamic and combinatorial control of gene expression by nuclear retinoic acid receptors (RARs)

    PubMed Central

    Rochette-Egly, Cécile; Germain, Pierre

    2009-01-01

    Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity. PMID:19471584

  6. Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

    PubMed

    Takeuchi, Jun; Okamoto, Masanori; Akiyama, Tomonori; Muto, Takuya; Yajima, Shunsuke; Sue, Masayuki; Seo, Mitsunori; Kanno, Yuri; Kamo, Tsunashi; Endo, Akira; Nambara, Eiji; Hirai, Nobuhiro; Ohnishi, Toshiyuki; Cutler, Sean R; Todoroki, Yasushi

    2014-06-01

    The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class. PMID:24792952

  7. Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

    PubMed

    Takeuchi, Jun; Okamoto, Masanori; Akiyama, Tomonori; Muto, Takuya; Yajima, Shunsuke; Sue, Masayuki; Seo, Mitsunori; Kanno, Yuri; Kamo, Tsunashi; Endo, Akira; Nambara, Eiji; Hirai, Nobuhiro; Ohnishi, Toshiyuki; Cutler, Sean R; Todoroki, Yasushi

    2014-06-01

    The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

  8. Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

    SciTech Connect

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae; E-mail: jwoo@isc.chubu.ac.jp

    2007-03-30

    Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-{kappa}B ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway.

  9. Retinoic Acid and LTP Recruit Postsynaptic AMPA-Receptors Using Distinct SNARE-Dependent Mechanisms

    PubMed Central

    Arendt, Kristin L.; Zhang, Yingsha; Jurado, Sandra; Malenka, Robert C.; Südhof, Thomas C.; Chen, Lu

    2015-01-01

    SUMMARY Retinoic acid- (RA-) dependent homeostatic plasticity and NMDA-receptor-dependent LTP, a form of Hebbian plasticity, both enhance synaptic strength by increasing the abundance of postsynaptic AMPA receptors (AMPARs). However, it is unclear whether the molecular mechanisms mediating AMPAR-trafficking during homeostatic and Hebbian plasticity differ, and unknown how RA-signaling impacts Hebbian plasticity. Here, we show that RA increases postsynaptic AMPAR-abundance by an activity-dependent mechanism that requires a unique SNARE-dependent fusion machinery different from that mediating LTP. Specifically, RA-induced AMPAR-trafficking did not involve complexin, which activates SNARE complexes containing syntaxin-1 or -3 but not complexes containing syntaxin-4, whereas LTP required complexin. Moreover, RA-induced AMPAR trafficking utilized the Q-SNARE syntaxin-4 whereas LTP utilized syntaxin-3; both additionally required the Q-SNARE SNAP-47 and the R-SNARE synatobrevin-2. Finally, acute RA treatment blocked subsequent LTP expression, probably by increasing AMPAR-trafficking. Thus, RA-induced homeostatic plasticity involves a novel, activity-dependent postsynaptic AMPAR-trafficking pathway mediated by a unique SNARE-dependent fusion machinery. PMID:25843403

  10. Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

    PubMed

    Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin; Urban, Sylvia; Osz, Judit; Chatagnon, Amandine; Delacroix, Laurence; Langer, Diana; Rochel, Natacha; Moras, Dino; Benoit, Gerard; Davidson, Irwin

    2012-07-27

    Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

  11. Hydroxycarboxylic acid receptor 2 mediates dimethyl fumarate’s protective effect in EAE

    PubMed Central

    Chen, Hui; Assmann, Julian C.; Krenz, Antje; Rahman, Mahbubur; Grimm, Myriam; Karsten, Christian M.; Köhl, Jörg; Offermanns, Stefan; Wettschureck, Nina; Schwaninger, Markus

    2014-01-01

    Taken orally, the drug dimethyl fumarate (DMF) has been shown to improve functional outcomes for patients with MS; however, it is unclear how DMF mediates a protective effect. DMF and, more so, its active metabolite, monomethyl fumarate, are known agonists of the hydroxycarboxylic acid receptor 2 (HCA2), a G protein–coupled membrane receptor. Here, we evaluated the contribution of HCA2 in mediating the protective effect afforded by DMF in EAE, a mouse model of MS. DMF treatment reduced neurological deficit, immune cell infiltration, and demyelination of the spinal cords in wild-type mice, but not in Hca2–/– mice, indicating that HCA2 is required for the therapeutic effect of DMF. In particular, DMF decreased the number of infiltrating neutrophils in a HCA2-dependent manner, likely by interfering with neutrophil adhesion to endothelial cells and chemotaxis. Together, our data indicate that HCA2 mediates the therapeutic effects of DMF in EAE. Furthermore, identification of HCA2 as a molecular target may help to optimize MS therapy. PMID:24691444

  12. Molecular regulation of lysophosphatidic acid receptor 1 trafficking to the cell surface.

    PubMed

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Dong, Su; Xiao, Shuqi; Zhao, Yutong

    2014-11-01

    The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84-87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation of Erk, and the cytokine release in response to LPA treatment. These results suggest that Ile325, Tyr85, and Leu87 within LPA1 are essential for LPA1 protein properly folding in the ER.

  13. Synaptic enhancement induced by gintonin via lysophosphatidic acid receptor activation in central synapses.

    PubMed

    Park, Hoyong; Kim, Sungmin; Rhee, Jeehae; Kim, Hyeon-Joong; Han, Jung-Soo; Nah, Seung-Yeol; Chung, ChiHye

    2015-03-01

    Lysophosphatidic acid (LPA) is one of the well-characterized, ubiquitous phospholipid molecules. LPA exerts its effect by activating G protein-coupled receptors known as LPA receptors (LPARs). So far, LPAR signaling has been critically implicated during early development stages, including the regulation of synapse formation and the morphology of cortical and hippocampal neurons. In adult brains, LPARs seem to participate in cognitive as well as emotional learning and memory. Recent studies using LPAR1-deficient mice reported impaired performances in a number of behavioral tasks, including the hippocampus-dependent spatial memory and fear conditioning tests. Nevertheless, the effect of LPAR activation in the synaptic transmission of central synapses after the completion of embryonic development has not been investigated. In this study, we took advantage of a novel extracellular agonist for LPARs called gintonin to activate LPARs in adult brain systems. Gintonin, a recently identified active ingredient in ginseng, has been shown to activate LPARs and mobilize Ca(2+) in an artificial cell system. We found that the activation of LPARs by application of gintonin acutely enhanced both excitatory and inhibitory transmission in central synapses, albeit through tentatively distinct mechanisms. Gintonin-mediated LPAR activation primarily resulted in synaptic enhancement and an increase in neuronal excitability in a phospholipase C-dependent manner. Our findings suggest that LPARs are able to directly potentiate synaptic transmission in central synapses when stimulated exogenously. Therefore, LPARs could serve as a useful target to modulate synaptic activity under pathological conditions, including neurodegenerative diseases.

  14. Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

    PubMed

    Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

    2016-09-01

    Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. PMID:27513962

  15. Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes

    PubMed Central

    Love, Crystal E.; Prince, Victoria E.

    2012-01-01

    Background The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. Results We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2 and nr2f5. These genes show highly regulated patterns of expression within the CNS, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and Fgf signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere-specific expression. Conclusions We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region. PMID:22836912

  16. Depletion of Retinoic Acid Receptors Initiates a Novel Positive Feedback Mechanism that Promotes Teratogenic Increases in Retinoic Acid

    PubMed Central

    D'Aniello, Enrico; Rydeen, Ariel B.; Anderson, Jane L.; Mandal, Amrita; Waxman, Joshua S.

    2013-01-01

    Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA. PMID:23990796

  17. Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan.

    PubMed

    Inoue, Daisuke; Nakama, Koki; Sawada, Kazuko; Watanabe, Taro; Takagi, Mai; Sei, Kazunari; Yang, Min; Hirotsuji, Junji; Hu, Jianying; Nishikawa, Jun-ichi; Nakanishi, Tsuyoshi; Ike, Michihiko

    2010-04-01

    This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARalpha agonist contamination in the river basins. RARalpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARalpha agonists are always present and widespread in the rivers. Comparative investigation of RARalpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARalpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARalpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARalpha agonists in the rivers. Although a trial conducted to identify RARalpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARalpha agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARalpha agonists with high

  18. Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

    PubMed

    Ayaori, Makoto; Yakushiji, Emi; Ogura, Masatsune; Nakaya, Kazuhiro; Hisada, Tetsuya; Uto-Kondo, Harumi; Takiguchi, Shunichi; Terao, Yoshio; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Yogo, Makiko; Uehara, Yoshinari; Kagechika, Hiroyuki; Nakanishi, Tsuyoshi; Ikewaki, Katsunori

    2012-04-01

    ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

  19. Effect of eicosapentaenoic acid on E-type prostaglandin synthesis and EP4 receptor signaling in human colorectal cancer cells.

    PubMed

    Hawcroft, Gillian; Loadman, Paul M; Belluzzi, Andrea; Hull, Mark A

    2010-08-01

    The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E(2) in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE(2) and generation of PGE(3). Alone, PGE(3) bound and activated the PGE(2) EP4 receptor but with reduced affinity and efficacy compared with its "natural" ligand PGE(2). However, in the presence of PGE(2), PGE(3) acted as an antagonist of EP4 receptor-dependent 3',5' cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent "PGE(2)-to-PGE(3) switch" in human CRC cells and that PGE(3) acts as a partial agonist at the PGE(2) EP4 receptor.

  20. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    PubMed Central

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease. PMID:26796668

  1. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  2. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  3. 2-Aryl(pyrrolidin-4-yl)acetic acids are potent agonists of sphingosine-1-phosphate (S1P) receptors.

    PubMed

    Yan, Lin; Budhu, Richard; Huo, Pei; Lynch, Christopher L; Hale, Jeffrey J; Mills, Sander G; Hajdu, Richard; Keohane, Carol A; Rosenbach, Mark J; Milligan, James A; Shei, Gan-Ju; Chrebet, Gary; Bergstrom, James; Card, Deborah; Mandala, Suzanne M

    2006-07-01

    A series of 2-aryl(pyrrolidin-4-yl)acetic acids were synthesized and their biological activities were evaluated as agonists of S1P receptors. These analogs were able to induce lowering of lymphocyte counts in the peripheral blood of mice and were found to have good overall pharmacokinetic properties in rat.

  4. Plant Lectin Can Target Receptors Containing Sialic Acid, Exemplified by Podoplanin, to Inhibit Transformed Cell Growth and Migration

    PubMed Central

    Shen, Yongquan; Acharya, Nimish K.; Han, Min; McNulty, Dean E.; Hasegawa, Hitoki; Hyodo, Toshinori; Senga, Takeshi; Geng, Jian-Guo; Kosciuk, Mary; Shin, Seung S.; Goydos, James S.; Temiakov, Dmitry; Nagele, Robert G.; Goldberg, Gary S.

    2012-01-01

    Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth. PMID:22844530

  5. Immunochemical demonstration that amino acids 360-377 of the acetylcholine receptor gamma-subunit are cytoplasmic

    PubMed Central

    1985-01-01

    Two monoclonal antibodies (mabs) previously prepared against Torpedo acetylcholine receptor are shown to recognize a synthetic nonadecapeptide corresponding to lys360-glu377 of the gamma subunit. The reaction was demonstrated by solid-phase enzyme-linked immunoabsorbent assays, by inhibition of binding of the mabs to receptor, and by immunoprecipitation of the peptide conjugated to bovine serum albumin. Immunogold electron microscopy on isolated postsynaptic membranes from Torpedo showed that both mabs bind to intracellular epitopes on the receptor. These results establish that amino acid residues 360-377 of the receptor gamma-subunit, and probably the analogous region of the delta-subunit, reside on the cytoplasmic side of the membrane. Since the primary structures of all four subunits suggest a common transmembrane arrangement, the corresponding domains of the alpha- and beta-subunits are probably also cytoplasmic. PMID:3972889

  6. Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro.

    PubMed

    Dietz, Birgit M; Mahady, Gail B; Pauli, Guido F; Farnsworth, Norman R

    2005-08-18

    Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABA(A) receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT(5a) receptor, but only weak binding affinity to the 5-HT(2b) and the serotonin transporter. Subsequent binding studies focused on the 5-HT(5a) receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep-wake cycle. The PE extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(5a) receptor (86% at 50 microg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC(50) curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC(50) of 15.7 ng/ml for the high-affinity state and 27.7 microg/ml for the low-affinity state. The addition of GTP (100 microM) resulted in a right-hand shift of the binding curve with an IC(50) of 11.4 microg/ml. Valerenic acid, the active constituent of both extracts, had an IC(50) of 17.2 microM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT(5a) receptor. PMID:15921820

  7. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival

    PubMed Central

    Rice, Dennis S.; Calandria, Jorgelina M.; Gordon, William C.; Jun, Bokkyoo; Zhou, Yongdong; Gelfman, Claire M.; Li, Songhua; Jin, Minghao; Knott, Eric J.; Chang, Bo; Abuin, Alex; Issa, Tawfik; Potter, David; Platt, Kenneth A.; Bazan, Nicolas G.

    2015-01-01

    The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity. PMID:25736573

  8. GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

    PubMed Central

    Gould, Timothy; Chen, Lixin; Emri, Zsuzsa; Pirttimaki, Tiina; Errington, Adam C.; Crunelli, Vincenzo; Parri, H. Rheinallt

    2014-01-01

    The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB. PMID:25225100

  9. Acidic phospholipids govern the enhanced activation of IgG-B cell receptor.

    PubMed

    Chen, Xiangjun; Pan, Weiling; Sui, Yinqiang; Li, Hua; Shi, Xiaoshan; Guo, Xingdong; Qi, Hai; Xu, Chenqi; Liu, Wanli

    2015-10-06

    B cells that express the isotype-switched IgG-B cell receptor (IgG-BCR) are one of the driving forces for antibody memory. To allow for a rapid memory IgG antibody response, IgG-BCR evolved into a highly effective signalling machine. Here, we report that the positively charged cytoplasmic domain of mIgG (mIgG-tail) specifically interacts with negatively charged acidic phospholipids. The key immunoglobulin tail tyrosine (ITT) in mIgG-tail is thus sequestered in the membrane hydrophobic core in quiescent B cells. Pre-disruption of such interaction leads to excessive recruitment of BCRs and inflated BCR signalling upon antigen stimulation, resulting in hyperproliferation of primary B cells. Physiologically, membrane-sequestered mIgG-tail can be released by antigen engagement or Ca(2+) mobilization in the initiation of B cell activation. Our studies suggest a novel regulatory mechanism for how dynamic association of mIgG-tail with acidic phospholipids governs the enhanced activation of IgG-BCR.

  10. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    PubMed Central

    Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

  11. Selective Retinoic Acid Receptor γ Agonists Promote Repair of Injured Skeletal Muscle in Mouse

    PubMed Central

    Di Rocco, Agnese; Uchibe, Kenta; Larmour, Colleen; Berger, Rebecca; Liu, Min; Barton, Elisabeth R.; Iwamoto, Masahiro

    2016-01-01

    Retinoic acid signaling regulates several biological events, including myogenesis. We previously found that retinoic acid receptor γ (RARγ) agonist blocks heterotopic ossification, a pathological bone formation that mostly occurs in the skeletal muscle. Interestingly, RARγ agonist also weakened deterioration of muscle architecture adjacent to the heterotopic ossification lesion, suggesting that RARγ agonist may oppose skeletal muscle damage. To test this hypothesis, we generated a critical defect in the tibialis anterior muscle of 7-week-old mice with a cautery, treated them with RARγ agonist or vehicle corn oil, and examined the effects of RARγ agonist on muscle repair. The muscle defects were partially repaired with newly regenerating muscle cells, but also filled with adipose and fibrous scar tissue in both RARγ-treated and control groups. The fibrous or adipose area was smaller in RARγ agonist–treated mice than in the control. In addition, muscle repair was remarkably delayed in RARγ-null mice in both critical defect and cardiotoxin injury models. Furthermore, we found a rapid increase in retinoid signaling in lacerated muscle, as monitored by retinoid signaling reporter mice. Together, our results indicate that endogenous RARγ signaling is involved in muscle repair and that selective RARγ agonists may be beneficial to promote repair in various types of muscle injuries. PMID:26205250

  12. Effects of retinoic acid receptor-selective agonists on human nasal epithelial cell differentiation.

    PubMed

    Million, K; Tournier, F; Houcine, O; Ancian, P; Reichert, U; Marano, F

    2001-12-01

    Retinoids play a critical role in the maintenance of the mucociliary phenotype of epithelial cells in the upper respiratory tract. To determine the role of retinoic acid receptors (RARs) in the regulation of epithelial differentiation, we tested the effect of the synthetic retinoids CD336, CD2019, and CD666, selective agonists for RARalpha, RARbeta, and RARgamma, respectively, during differentiation of human nasal epithelial (HNE) cells in vitro. Using glutamylated tubulin and transglutaminase I (Tg I) as markers of ciliated cell and squamous cell differentiation, respectively, we showed that retinoic acid (RA) stimulated mucociliary differentiation and, in parallel, inhibited squamous cell differentiation. The agonists of the three RARs independently induced ciliogenesis and inhibited squamous cell differentiation by downregulating Tg I expression in a dose- and time-dependent manner. Antagonists specific for the three RARs abolished the effects of the corresponding agonists, demonstrating an RAR-specific mediated effect. Moreover, treatment of retinoid-deficient cultures with RAR agonists induced conversion of the squamous-like phenotype into a ciliated phenotype. In conclusion, all three RARs are potentially involved in the differentiating effects of RA in respiratory epithelial cells.

  13. Succinate reverses in-vitro platelet inhibition by acetylsalicylic acid and P2Y receptor antagonists.

    PubMed

    Spath, Brigitte; Hansen, Arne; Bokemeyer, Carsten; Langer, Florian

    2012-01-01

    High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000 µM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100 µM succinate. Similarly, succinate completely (at 1000 µM) or partially (at 100 µM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P

  14. Allosteric regulation of the discriminative responsiveness of retinoic acid receptor to natural and synthetic ligands by retinoid X receptor and DNA.

    PubMed

    Mouchon, A; Delmotte, M H; Formstecher, P; Lefebvre, P

    1999-04-01

    Transcriptional activation by retinoids is mediated through two families of nuclear receptors, all-trans-retinoic acid (RARs) and 9-cis retinoic acid receptors (RXRs). Conformationally restricted retinoids are used to achieve selective activation of RAR isotype alpha, beta or gamma, which reduces side effects in therapeutical applications. Synthetic retinoids mimic some of all-trans retinoic acid biological effects in vivo but interact differently with the ligand binding domain of RARalpha and induce distinct structural transitions of the receptor. In this report, we demonstrate that RAR-selective ligands have distinct quantitative activation properties which are reflected by their abilities to promote interaction of DNA-bound human RXRalpha (hRXRalpha)-hRARalpha heterodimers with the nuclear receptor coactivator (NCoA) SRC-1 in vitro. The hormone response element core motifs spacing defined the relative affinity of liganded heterodimers for two NCoAs, SRC-1 and RIP140. hRXRalpha activating function 2 was critical to confer hRARalpha full responsiveness but not differential sensitivity of hRARalpha to natural or synthetic retinoids. We also provide evidence showing that lysines located in helices 3 and 4, which define part of hRARalpha NCoA binding surface, contribute differently to (i) the transcriptional activity and (ii) the interaction of RXR-RAR heterodimers with SRC-1, when challenged by either natural or RAR-selective retinoids. Thus, ligand structure, DNA, and RXR exert allosteric regulations on hRARalpha conformation organized as a DNA-bound heterodimer. We suggest that the use of physically distinct NCoA binding interfaces may be important in controlling specific genes by conformationally restricted ligands.

  15. Amino Acid Derivatives as Bitter Taste Receptor (T2R) Blockers*

    PubMed Central

    Pydi, Sai P.; Sobotkiewicz, Tyler; Billakanti, Rohini; Bhullar, Rajinder P.; Loewen, Michele C.; Chelikani, Prashen

    2014-01-01

    In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-l-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 μm and 59 ± 18 nm, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues. PMID:25059668

  16. Porcine Sapelovirus Uses α2,3-Linked Sialic Acid on GD1a Ganglioside as a Receptor

    PubMed Central

    Kim, Deok-Song; Son, Kyu-Yeol; Koo, Kyung-Min; Kim, Ji-Yun; Alfajaro, Mia Madel; Park, Jun-Gyu; Hosmillo, Myra; Soliman, Mahmoud; Baek, Yeong-Bin; Cho, Eun-Hyo; Lee, Ju-Hwan; Kang, Mun-Il

    2016-01-01

    ABSTRACT The receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6′-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensis lectin and Sambucus nigra lectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy. IMPORTANCE The porcine sapelovirus (PSV) is known to cause enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. However, the receptor(s) that the PSV utilizes to enter host cells remains largely unknown. Using a variety of approaches, we showed that α2,3-linked terminal sialic acid (SA) on the cell surface GD1a ganglioside could be used for PSV

  17. Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

    PubMed Central

    Samways, Damien S.K.; Egan, Terrance M.

    2007-01-01

    P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors. PMID:17325195

  18. Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique.

    PubMed

    Ramakrishnan, Latha; Hess, George P

    2005-06-14

    The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.

  19. Actions of Xanthurenic acid, a putative endogenous Group II metabotropic glutamate receptor agonist, on sensory transmission in the thalamus.

    PubMed

    Copeland, C S; Neale, S A; Salt, T E

    2013-03-01

    Xanthurenic acid (XA), a molecule arising from tryptophan metabolism by transamination of 3-hydroxykynurenine, has recently been identified as an endogenous Group II (mGlu2 and mGlu3) metabotropic glutamate (mGlu) receptor ligand in vitro. Impairments in Group II mGlu receptor expression and function have been implicated in the pathophysiology of schizophrenia, as have multiple steps in the kynurenine metabolism pathway. Therefore, we examined XA in vivo to further investigate its potential as a Group II mGlu receptor ligand using a preparation that has been previously demonstrated to efficiently reveal the action of other Group II mGlu receptor ligands in vivo. Extracellular single-neurone recordings were made in the rat ventrobasal thalamus (VB) in conjunction with iontophoresis of agonists, an antagonist and a positive allosteric modulator and/or intravenous (i.v.) injection of XA. We found the XA effect on sensory inhibition, when applied iontophoretically and i.v., was similar to that of other Group II mGlu receptor agonists in reducing inhibition evoked in the VB from the thalamic reticular nucleus upon physiological sensory stimulation. Furthermore, we postulate that XA may be the first potential endogenous allosteric agonist (termed 'endocoid') for the mGlu receptors. As the Group II receptors and kynurenine metabolism pathway have both been heavily implicated in the pathophysiology of schizophrenia, XA could play a pivotal role in antipsychotic research as this potential endocoid represents both a convergence within these two biological parameters and a novel class of Group II mGlu receptor ligand. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22491023

  20. Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect.

    PubMed

    Nowickyj, Shaun M; Chithalen, James V; Cameron, Don; Tyshenko, Michael G; Petkovich, Martin; Wyatt, Gerard R; Jones, Glenville; Walker, Virginia K

    2008-07-15

    The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC(50) = 61.2-107.7 nM; K(d) = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.

  1. Induction of intermediate mesoderm by retinoic acid receptor signaling from differentiating mouse embryonic stem cells.

    PubMed

    Oeda, Shiho; Hayashi, Yohei; Chan, Techuan; Takasato, Minoru; Aihara, Yuko; Okabayashi, Koji; Ohnuma, Kiyoshi; Asashima, Makoto

    2013-01-01

    Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

  2. A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid.

    PubMed

    Wanner, Kevin W; Nichols, Andrew S; Walden, Kimberly K O; Brockmann, Axel; Luetje, Charles W; Robertson, Hugh M

    2007-09-01

    By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.

  3. Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

    PubMed Central

    MIYABE, Masahiro; GIN, Azusa; ONOZAWA, Eri; DAIMON, Mana; YAMADA, Hana; ODA, Hitomi; MORI, Akihiro; MOMOTA, Yutaka; AZAKAMI, Daigo; YAMAMOTO, Ichiro; MOCHIZUKI, Mariko; SAKO, Toshinori; TAMURA, Katsutoshi; ISHIOKA, Katsumi

    2015-01-01

    G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs. PMID:25960032

  4. Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis

    PubMed Central

    Hov, Johannes R.; Keitel, Verena; Laerdahl, Jon K.; Spomer, Lina; Ellinghaus, Eva; ElSharawy, Abdou; Melum, Espen; Boberg, Kirsten M.; Manke, Thomas; Balschun, Tobias; Schramm, Christoph; Bergquist, Annika; Weismüller, Tobias; Gotthardt, Daniel; Rust, Christian; Henckaerts, Liesbet; Onnie, Clive M.; Weersma, Rinse K.; Sterneck, Martina; Teufel, Andreas; Runz, Heiko; Stiehl, Adolf; Ponsioen, Cyriel Y.; Wijmenga, Cisca; Vatn, Morten H.; Stokkers, Pieter C. F.; Vermeire, Severine; Mathew, Christopher G.; Lie, Benedicte A.; Beuers, Ulrich; Manns, Michael P.; Schreiber, Stefan; Schrumpf, Erik; Häussinger, Dieter; Franke, Andre; Karlsen, Tom H.

    2010-01-01

    Background TGR5, the G protein-coupled bile acid receptor 1 (GPBAR1), has been linked to inflammatory pathways as well as bile homeostasis, and could therefore be involved in primary sclerosing cholangitis (PSC) a chronic inflammatory bile duct disease. We aimed to extensively investigate TGR5 sequence variation in PSC, as well as functionally characterize detected variants. Methodology/Principal Findings Complete resequencing of TGR5 was performed in 267 PSC patients and 274 healthy controls. Six nonsynonymous mutations were identified in addition to 16 other novel single-nucleotide polymorphisms. To investigate the impact from the nonsynonymous variants on TGR5, we created a receptor model, and introduced mutated TGR5 constructs into human epithelial cell lines. By using confocal microscopy, flow cytometry and a cAMP-sensitive luciferase assay, five of the nonsynonymous mutations (W83R, V178M, A217P, S272G and Q296X) were found to reduce or abolish TGR5 function. Fine-mapping of the previously reported PSC and UC associated locus at chromosome 2q35 in large patient panels revealed an overall association between the TGR5 single-nucleotide polymorphism rs11554825 and PSC (odds ratio  = 1.14, 95% confidence interval: 1.03–1.26, p = 0.010) and UC (odds ratio  = 1.19, 95% confidence interval 1.11–1.27, p = 8.5×10−7), but strong linkage disequilibrium precluded demarcation of TGR5 from neighboring genes. Conclusions/Significance Resequencing of TGR5 along with functional investigations of novel variants provided unique insight into an important candidate gene for several inflammatory and metabolic conditions. While significant TGR5 associations were detected in both UC and PSC, further studies are needed to conclusively define the role of TGR5 variation in these diseases. PMID:20811628

  5. Central phencyclidine (PCP) receptor binding is glutamate dependent: evidence for a PCP/excitatory amino acid receptor (EAAR) complex

    SciTech Connect

    Loo, P.; Braunwalder, A.; Lehmann, J.; Williams, M.

    1986-03-01

    PCP and other dissociative anesthetica block the increase in neuronal firing rate evoked by the EAAR agonist, N-methyl-Daspartate. NMDA and other EAAs such as glutamate (glu) have not been previously shown to affect PCP ligand binding. In the present study, using once washed rat forebrain membranes, 10 ..mu..M-glu was found to increase the binding of (/sup 3/H)TCP, a PCP analog, to defined PCP recognition sites by 20%. Removal of glu and aspartate (asp) by extensive washing decreased TCP binding by 75-90%. In these membranes, 10 ..mu..M L-glu increased TCP binding 3-fold. This effect was stereospecific and evoked by other EAAs with the order of activity, L-glu > D-asp > L- asp > NMDA > D-glu > quisqualate. Kainate, GABA, NE, DA, 5-HT, 2-chloroadenosine, oxotremorine and histamine had no effect on TCP binding at concentrations up to 100 ..mu..M. The effects of L-glu were attenuated by the NMDA-type receptor antagonist, 2-amino-7--phosphonoheptanoate (AP7; 10 ..mu..M-1 mM). These findings indicate that EAAS facilitate TCP binding, possibly through NMDA-type receptors. The observed interaction between the PCP receptor and EAARs may reflect the existence of a macromolecular receptor complex similar to that demonstrated for the benzodiazepines and GABA.

  6. Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms.

    PubMed

    Yao, Pei-Li; Chen, Li Ping; Dobrzański, Tomasz P; Phillips, Dylan A; Zhu, Bokai; Kang, Boo-Hyon; Gonzalez, Frank J; Peters, Jeffrey M

    2015-11-01

    Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARβ/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARβ/δ. Expression of PPARβ/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARβ/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARβ/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARβ/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARβ/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

  7. Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus.

    PubMed

    Cardona-Gómez, Gloria Patricia; Arango-Davila, Cesar; Gallego-Gómez, Juan Carlos; Barrera-Ocampo, Alvaro; Pimienta, Hernan; Garcia-Segura, Luis Miguel

    2006-08-21

    During cerebral ischemia, part of the damage associated with the hyperactivation of glutamate receptors results from the hyperphosphorylation of the microtubule-associated protein Tau. Previous studies have shown that estradiol treatment reduces neural damage after cerebral ischemia. Here, we show that transient occlusion of the middle cerebral artery results in the hyperphosphorylation of Tau and in a significant increase in the association of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type glutamate receptor subunits 2/3 in the hippocampus. Estradiol treatment decreased hippocampal injury, inhibited glycogen synthase kinase-3beta and decreased the hyperphosphorylation of Tau and the interaction of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. These findings suggest that ischemia produces a strong association between Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, and estradiol can exert at least part of its neuroprotective activity through inhibition of glycogen synthase kinase-3beta.

  8. Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor

    PubMed Central

    Chennupati, Ramesh; Nüsing, Rolf M.; Offermanns, Stefan

    2016-01-01

    19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists. PMID:27662627

  9. A putative G-protein-coupled receptor, H218, is down-regulated during the retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

    PubMed

    Li, Y; MacLennan, A J; Rogers, M B

    1998-03-15

    We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor beta or gamma signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.

  10. PERFLUOROPHOSPHONIC ACID ACTIVATES PEROXISOME PROUFERATOR-ACTIVATED RECEPTOR-ALPHA BUT NOT CONSTITUTIVE ANDROSTANE RECEPTOR IN THE MURINE LIVER

    EPA Science Inventory

    Masurf FS-780 is a commercial perfluoro-chemical mixture that contains C612-perfluoroalkylphosphonic acid (PFPA) derivatives. PFPAs have received recent attention as a previously under recognized subclass of perfluoroalkyl acids (PFAAs) that are found in the environment. The curr...

  11. Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation

    PubMed Central

    Mosquna, Assaf; Peterson, Francis C.; Park, Sang-Youl; Lozano-Juste, Jorge; Volkman, Brian F.; Cutler, Sean R.

    2011-01-01

    Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta. PMID:22139369

  12. Bile acid regulates c-Jun expression through the orphan nuclear receptor SHP induction in gastric cells

    SciTech Connect

    Park, Won Il; Park, Min Jung; An, Jin Kwang; Choi, Yung Hyun; Kim, Hye Young; Cheong, JaeHun Yang, Ung Suk

    2008-05-02

    Bile reflux is considered to be one of the most important causative factors in gastric carcinogenesis, due to the attendant inflammatory changes in the gastric mucosa. In this study, we have assessed the molecular mechanisms inherent to the contribution of bile acid to the transcriptional regulation of inflammatory-related genes. In this study, we demonstrated that bile acid induced the expression of the SHP orphan nuclear receptor at the transcriptional level via c-Jun activation. Bile acid also enhanced the protein interaction of NF-{kappa}B and SHP, thereby resulting in an increase in c-Jun expression and the production of the inflammatory cytokine, TNF{alpha}. These results indicate that bile acid performs a critical function in the regulation of the induction of inflammatory-related genes in gastric cells, and that bile acid-mediated gene expression provides a pre-clue for the development of gastric cellular malformation.

  13. Blockade of lysophosphatidic acid receptors LPAR1/3 ameliorates lung fibrosis induced by irradiation

    SciTech Connect

    Gan, Lu; Xue, Jian-Xin; Li, Xin; Liu, De-Song; Ge, Yan; Ni, Pei-Yan; Deng, Lin; Lu, You; Jiang, Wei

    2011-05-27

    Highlights: {yields} Lysophosphatidic acid (LPA) levels and its receptors LPAR1/3 transcripts were elevated during the development of radiation-induced lung fibrosis. {yields} Lung fibrosis was obviously alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. {yields} VPC12249 administration effectively inhibited radiation-induced fibroblast accumulation in vivo, and suppressed LPA-induced fibroblast proliferation in vitro. {yields} LPA-LPAR1/3 signaling regulated TGF{beta}1 and CTGF expressions in radiation-challenged lungs, but only influenced CTGF expression in cultured fibroblasts. {yields} LPA-LPAR1/3 signaling induced fibroblast proliferation through a CTGF-dependent pathway, rather than through TGF{beta}1 activation. -- Abstract: Lung fibrosis is a common and serious complication of radiation therapy for lung cancer, for which there are no efficient treatments. Emerging evidence indicates that lysophosphatidic acid (LPA) and its receptors (LPARs) are involved in the pathogenesis of fibrosis. Here, we reported that thoracic radiation with 16 Gy in mice induced development of radiation lung fibrosis (RLF) accompanied by obvious increases in LPA release and LPAR1 and LPAR3 (LPAR1/3) transcripts. RLF was significantly alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. VPC12249 administration effectively prolonged animal survival, restored lung structure, inhibited fibroblast accumulation and reduced collagen deposition. Moreover, profibrotic cytokines in radiation-challenged lungs obviously decreased following administration of VPC12249, including transforming growth factor {beta}1 (TGF{beta}1) and connective tissue growth factor (CTGF). In vitro, LPA induced both fibroblast proliferation and CTGF expression in a dose-dependent manner, and both were suppressed by blockade of LPAR1/3. The pro-proliferative activity of LPA on fibroblasts was inhibited by siRNA directed against CTGF. Together, our data suggest that the LPA-LPAR1

  14. Dominant negative mutant of retinoic acid receptor alpha inhibits retinoic acid-induced P19 cell differentiation by binding to DNA.

    PubMed

    Costa, S L; McBurney, M W

    1996-05-25

    Retinoic acid (RA) is a potent inducer of P19 cell differentiation. RA activity is thought to be mediated by nuclear RA receptors (RARs), transcription factors whose activity is dependent on RA. There are three RARs called alpha, beta, and gamma. We created truncated versions of the three RARs and compared their activities as inhibitors of RA-mediated gene transcription and of P19 cell differentiation. Only mutants of the RAR alpha were inhibitory in these assays. A mutant of RAR alpha carrying a 10-amino-acid insert was able to heterodimerize with RXRbeta or with the normal RAR alpha and the inhibitory activity of this mutant was dependent on an intact DNA binding domain. We conclude that dominant negative mutants of RAR alpha act by heterodimerizing with RXRs or RARs and binding to RA response elements on DNA, thereby preventing binding of the normal receptors to those sites. PMID:8635515

  15. Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids

    PubMed Central

    2015-01-01

    We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide–alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism. PMID:25524496

  16. Trans Fatty Acid Derived Phospholipids Show Increased Membrane Cholesterol and Reduced Receptor Activation as Compared to Their Cis Analogs

    PubMed Central

    Niu, Shui-Lin; Mitchell, Drake C.; Litman, Burton J.

    2005-01-01

    The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid–cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid–receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA–phospholipid could be factors contributing to the elevation of LDL cholesterol. PMID:15766276

  17. Increasing human Th17 differentiation through activation of orphan nuclear receptor retinoid acid-related orphan receptor γ (RORγ) by a class of aryl amide compounds.

    PubMed

    Zhang, Wei; Zhang, Jing; Fang, Leiping; Zhou, Ling; Wang, Shuai; Xiang, Zhijun; Li, Yuan; Wisely, Bruce; Zhang, Guifeng; An, Gang; Wang, Yonghui; Leung, Stewart; Zhong, Zhong

    2012-10-01

    In a screen for small-molecule inhibitors of retinoid acid-related orphan receptor γ (RORγ), we fortuitously discovered that a class of aryl amide compounds behaved as functional activators of the interleukin 17 (IL-17) reporter in Jurkat cells. Three of these compounds were selected for further analysis and found to activate the IL-17 reporter with potencies of ∼0.1 μM measured by EC₅₀. These compounds were shown to directly bind to RORγ by circular dichroism-based thermal stability experiments. Furthermore, they can enhance an in vitro Th17 differentiation process in human primary T cells. As RORγ remains an orphan nuclear receptor, discovery of these aryl amide compounds as functional agonists will now provide pharmacological tools for us to dissect functions of RORγ and facilitate drug discovery efforts for immune-modulating therapies.

  18. Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element

    PubMed Central

    Claudel, Thierry; Sturm, Ekkehard; Duez, Hélène; Torra, Inés Pineda; Sirvent, Audrey; Kosykh, Vladimir; Fruchart, Jean-Charles; Dallongeville, Jean; Hum, Dean W.; Kuipers, Folkert; Staels, Bart

    2002-01-01

    Serum levels of HDL are inversely correlated with the risk of coronary heart disease. The anti-atherogenic effect of HDL is partially mediated by its major protein constituent apoA-I. In this study, we identify bile acids that are activators of the nuclear receptor farnesoid X receptor (FXR) as negative regulators of human apoA-I expression. Intrahepatocellular accumulation of bile acids, as seen in patients with progressive familial intrahepatic cholestasis and biliary atresia, was associated with diminished apoA-I serum levels. In human apoA-I transgenic mice, treatment with the FXR agonist taurocholic acid strongly decreased serum concentrations and liver mRNA levels of human apoA-I, which was associated with reduced serum HDL levels. Incubation of human primary hepatocytes and hepatoblastoma HepG2 cells with bile acids resulted in a dose-dependent downregulation of apoA-I expression. Promoter mutation analysis and gel-shift experiments in HepG2 cells demonstrated that bile acid–activated FXR decreases human apoA-I promoter activity by a negative FXR response element mapped to the C site. FXR bound this site and repressed transcription in a manner independent of retinoid X receptor. The nonsteroidal synthetic FXR agonist GW4064 likewise decreased apoA-I mRNA levels and promoter activity in HepG2 cells. PMID:11927623

  19. Effect of paraoxon on muscarinic, dopamine and. gamma. -aminobutyric acid receptors of brain and sensitivity to muscarinic antagonists

    SciTech Connect

    Fernando, J.C.R.; Hoskins, B.; Ho, I.K.

    1986-03-05

    Several acetylcholinesterase (AChE) inhibitors decrease muscarinic cholinergic (mACh) receptors in the brain, alteration of dopamine (DA) and ..gamma..-aminobutyric acid (GABA) receptors after AChE inhibition was also reported. In view of the important interactions among DA, GABA and ACh systems, whether this is a common effect of AChE inhibitors should be established. They report the effect of the AChE inhibitor, paraoxon, on DA, GABA and mACh receptors in the rat. The binding of /sup 3/H-QNB (for mACh), /sup 3/H-spiperone (for DA) and /sup 3/H-muscimol (for GABA) to striatal and hippocampal membranes was analyzed. Also, behavioral sensitivity to atropine was studied. Twenty-four hr after a single dose (0.75 mg/kg, s.c.) of paraoxon, the density of mACh receptors in the striatum was decreased but, at 3 days, no change was seen. In the hippocampus, the mACh receptors were not affected. Repeated treatment with paraoxon (0.3 mg/kg, 48 hourly) for 2 weeks reduced the mACh receptor density in both regions. Neither single nor repeated paraoxon treatment had an effect on DA or GABA receptors. After single or repeated dosing with paraoxon, myoclonus induced by atropine (10 mg/kg, i.p.) was enhanced. The results show rapid downregulation of mACh receptors by paraoxon. DA or GABA, however, appear not to be affected under these treatment regimens.

  20. Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Yang, Eun-Ju; Song, Kyung-Sik; Bae, Jong-Sup

    2013-09-01

    The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.

  1. Deletion of lysophosphatidic acid receptor LPA1 reduces neurogenesis in the mouse dentate gyrus

    PubMed Central

    Matas-Rico, Elisa; García-Diaz, Beatriz; Llebrez-Zayas, Pedro; López-Barroso, Diana; Santín, Luis; Pedraza, Carmen; Smith-Fernández, Anibal; Fernández-Llebrez, Pedro; Tellez, Teresa; Redondo; Chun, Jerold; De Fonseca, Fernando Rodríguez; Estivill-Torrús, Guillermo

    2013-01-01

    Neurogenesis persists in certain regions of the adult brain including the subgranular zone of the hippocampal dentate gyrus wherein its regulation is essential, particularly in relation to learning, stress and modulation of mood. Lysophosphatidic acid (LPA) is an extracellular signaling phospholipid with important neural regulatory properties mediated by specific G protein-coupled receptors, LPA1-5. LPA1 is highly expressed in the developing neurogenic ventricular zone wherein it is required for normal embryonic neurogenesis, and, by extension may play a role in adult neurogenesis as well. By means of the analyses of a variant of the original LPA1-null mutant mouse, termed the Malaga variant or “maLPA1-null,” which has recently been reported to have defective neurogenesis within the embryonic cerebral cortex, we report here a role for LPA1 in adult hippocampal neurogenesis. Proliferation, differentiation and survival of newly formed neurons are defective in the absence of LPA1 under normal conditions and following exposure to enriched environment and voluntary exercise. Furthermore, analysis of trophic factors in maLPA1-null mice demonstrated alterations in brain-derived neurotrophic factor and insulin growth factor 1 levels after enrichment and exercise. Morphological analyses of doublecortin positive cells revealed the anomalous prevalence of bipolar cells in the subgranular zone, supporting the operation of LPA1 signaling pathways in normal proliferation, maturation and differentiation of neuronal precursors. PMID:18708146

  2. N-Methyl D-Aspartate Receptor Antagonist Kynurenic Acid Affects Human Cortical Development

    PubMed Central

    Bagasrawala, Inseyah; Zecevic, Nada; Radonjić, Nevena V.

    2016-01-01

    Kynurenic acid (KYNA), a neuroactive metabolite of tryptophan degradation, acts as an endogenous N-methyl-D-aspartate receptor (NMDAR) antagonist. Elevated levels of KYNA have been observed in pregnant women after viral infections and are considered to play a role in neurodevelopmental disorders. However, the consequences of KYNA-induced NMDAR blockade in human cortical development still remain elusive. To study the potential impact of KYNA on human neurodevelopment, we used an in vitro system of multipotent cortical progenitors, i.e., radial glia cells (RGCs), enriched from human cerebral cortex at mid-gestation (16–19 gestational weeks). KYNA treatment significantly decreased RGCs proliferation and survival by antagonizing NMDAR. This alteration resulted in a reduced number of cortical progenitors and neurons while number and activation of astrocytes increased. KYNA treatment reduced differentiation of RGCs into GABAergic neurons, while differentiation into glutamatergic neurons was relatively spared. Furthermore, in mixed cortical cultures KYNA triggered an inflammatory response as evidenced by increased levels of the pro-inflammatory cytokine IL-6. In conclusion, elevated levels of KYNA play a significant role in human RGC fate determination by antagonizing NMDARs and by activating an inflammatory response. The altered cell composition observed in cell culture following exposure to elevated KYNA levels suggests a mechanism for impairment of cortical circuitry formation in the fetal brain after viral infection, as seen in neurodevelopmental disorders such as schizophrenia. PMID:27746712

  3. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

  4. Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

    PubMed

    Tsukahara, Tamotsu; Tsukahara, Ryoko; Fujiwara, Yuko; Yue, Junming; Cheng, Yunhui; Guo, Huazhang; Bolen, Alyssa; Zhang, Chunxiang; Balazs, Louisa; Re, Fabio; Du, Guangwei; Frohman, Michael A; Baker, Daniel L; Parrill, Abby L; Uchiyama, Ayako; Kobayashi, Tetsuyuki; Murakami-Murofushi, Kimiko; Tigyi, Gabor

    2010-08-13

    Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists. PMID:20705243

  5. The role of the C-terminus of the human hydroxycarboxylic acid receptors 2 and 3 in G protein activation using Gα-engineered yeast cells.

    PubMed

    Liu, Rongfang; van Veldhoven, Jacobus P D; IJzerman, Adriaan P

    2016-01-01

    In the present study we focused our attention on the family of hydroxycarboxylic acid (HCA) receptors, a GPCR family of three members, of which the HCA2 and HCA3 receptors share 95% high sequence identity but differ considerably in C-terminus length with HCA3 having the longest tail. The two receptors were expressed and analysed for their activation profile in Saccharomyces cerevisiae MMY yeast strains that have different G protein Gα subunits. The hHCA2 receptor was promiscuous in its G protein coupling preference. In the presence of nicotinic acid the hHCA2 receptor activated almost all G protein pathways except Gαq (MMY14). However, the Gα protein coupling profile of the hHCA3 receptor was less promiscuous, as the receptor only activated Gαi1 (MMY23) and Gαi3 (MMY24) pathways. We then constructed two mutant receptors by 'swapping' the short (HCA2) and long (HCA3) C-terminus. The differences in HCA2 and HCA3 receptor activation and G protein selectivity were not controlled, however, by their C-terminal tails, as we observed only minor differences between mutant and corresponding wild-type receptor. This study provides new insights into the G protein coupling profiles of the HCA receptors and the function of the receptor's C terminus, which may be extended to other GPCRs.

  6. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives

    PubMed Central

    Hintersteiner, J.; Luger, D.; Haider, M.; Pototschnig, G.; Mihovilovic, M. D.; Schwarzer, C.; Hering, S.

    2016-01-01

    Valerenic acid (VA)—a β2/3-selective GABA type A (GABAA) receptor modulator—displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA’s methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg

  7. Differential regulation of bile acid and cholesterol metabolism by the farnesoid X receptor in Ldlr -/- mice versus hamsters.

    PubMed

    Gardès, Christophe; Chaput, Evelyne; Staempfli, Andreas; Blum, Denise; Richter, Hans; Benson, G Martin

    2013-05-01

    Modulating bile acid synthesis has long been considered a good strategy by which to improve cholesterol homeostasis in humans. The farnesoid X receptor (FXR), the key regulator of bile acid synthesis, was, therefore, identified as an interesting target for drug discovery. We compared the effect of four, structurally unrelated, synthetic FXR agonists in two fat-fed rodent species and observed that the three most potent and selective agonists decreased plasma cholesterol in LDL receptor-deficient (Ldlr (-/-)) mice, but none did so in hamsters. Detailed investigation revealed increases in the expression of small heterodimer partner (Shp) in their livers and of intestinal fibroblast growth factor 15 or 19 (Fgf15/19) in mice only. Cyp7a1 expression and fecal bile acid (BA) excretion were strongly reduced in mice and hamsters by all four FXR agonists, whereas bile acid pool sizes were reduced in both species by all but the X-Ceptor compound in hamsters. In Ldlr (-/-) mice, the predominant bile acid changed from cholate to the more hydrophilic β-muricholate due to a strong repression of Cyp8b1 and increase in Cyp3a11 expression. However, FXR agonists caused only minor changes in the expression of Cyp8b1 and in bile acid profiles in hamsters. In summary, FXR agonist-induced decreases in bile acid pool size and lipophilicity and in cholesterol absorption and synthesis could explain the decreased plasma cholesterol in Ldlr (-/-) mice. In hamsters, FXR agonists reduced bile acid pool size to a smaller extent with minor changes in bile acid profile and reductions in sterol absorption, and consequently, plasma cholesterol was unchanged.

  8. The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing.

    PubMed

    Mun, Hee-Chang; Franks, Alison H; Culverston, Emma L; Krapcho, Karen; Nemeth, Edward F; Conigrave, Arthur D

    2004-12-10

    We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

  9. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    PubMed

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  10. Capsaicin, acid and heat-evoked currents in rat trigeminal ganglion neurons: relationship to functional VR1 receptors.

    PubMed

    Liu, L; Simon, S A

    2000-05-01

    Activation of primary trigeminal (TG) neurons by protons, capsaicin, or heat can evoke a variety of sensations, including tingling, stinging, warmth, and burning. Capsaicin and acid are trigeminal stimulants that are important in gustatory physiology. These stimuli can activate H(+)-gated ion channels and heterologously expressed VR1 receptors (vanilloid receptor 1). We have obtained evidence by using electrophysiological and pharmacological measurements on TG neurons that these three stimuli can activate many receptors, and we have determined the extent they behave similarly to VR1 receptors and H(+)-gated channels from the DEGenerin/ENaC superfamily. Whole-cell recordings from rat TG neurons revealed that protons evoked transient (Tp), sustained (Sp), and biphasic (TSp) currents. Tp currents had reversal potentials (Vr) of 24-45 mV, a pH(0.5) range from 5.5 to 6.5, and were inhibited by amiloride, suggesting the presence of functional H(+)-gated channels. Sp currents were inhibited by the VR1 antagonist capsazepine, had Vr's approximately 0 mV, and had pH(0.5) = 6.4. Capsaicin also activated transient (Tc), sustained (Sc), and biphasic (TSc) currents. At pH 5.9, the sensitivity of the Sc currents increased by about a factor of 10, which may partially account for the synergistic responses of acid in foods containing capsaicin. Heating TG neurons evoked a thermally active, capsazepine-inhibitable current with threshold temperature of 43 degrees C and Vr = 5 mV that is also present in neurons activated by and protons (Sp) and capsaicin (Sc). These data suggest that TG neurons have functional receptors that behave similarly to VR1. Activation of such receptors should result in a burning sensation, whereas activation of the transient and biphasic currents should result in other taste descriptors.

  11. Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

    PubMed Central

    Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

    1996-01-01

    OBJECTIVE: The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. SUMMARY BACKGROUND DATA: Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. METHODS: Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. RESULTS: Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. CONCLUSIONS: Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL

  12. Does smoking interfere with the effect of histamine H2-receptor antagonists on intragastric acidity in man?

    PubMed Central

    Bauerfeind, P; Cilluffo, T; Fimmel, C J; Emde, C; von Ritter, C; Kohler, W; Gugler, R; Gasser, T; Blum, A L

    1987-01-01

    The interaction between smoking and the effect of histamine H2-antagonists on intragastric acidity was examined in a double blind double dummy placebo controlled study. Healthy volunteers, 11 smokers and 10 non-smokers, were given, on four separate days at least one week apart, either placebo or cimetidine 800 mg nocte or ranitidine 2 X 150 mg per day or ranitidine 300 mg nocte. Tablets were taken at 2115 and 0900 h. Smokers smoked a cigarette hourly from 0700 to 2300 h. Breakfast, lunch, and dinner were standardised. Intragastric acidity was measured with a combined intragastric glass electrode and a solid state recorder. The subjects were fully ambulatory. The three histamine H2-receptor antagonist regimens were less effective (p = 0.04) in smokers than in non-smokers, but the difference between acidity of smokers and non-smokers was small. Means of medians of pH during a 24-h period with placebo, cimetidine 800 mg, ranitidine 2 X 150 mg and ranitidine 300 mg were 1.6, 2.3, 3.1, and 2.7 in smokers and 1.5, 2.7, 3.2, and 3.1 in non-smokers, respectively. In a second part of the study seven chronic smokers were reexamined after acutely stopping smoking: inhibition of gastric acidity by histamine H2-receptor antagonists was similar before and after withdrawal. Smoking does not affect intragastric acidity in untreated volunteers and only slightly decreases the effectiveness of histamine H2-receptor antagonists on intragastric acidity. This effect best in part explains the unfavourable effect of smoking on healing of peptic ulcer in patients treated with these drugs. PMID:3596336

  13. In vivo imaging of farnesoid X receptor activity reveals the ileum as the primary bile acid signaling tissue.

    PubMed

    Houten, Sander M; Volle, David H; Cummins, Carolyn L; Mangelsdorf, David J; Auwerx, Johan

    2007-06-01

    We generated and characterized a firefly luciferase reporter mouse for the nuclear receptor farnesoid X receptor (FXR). This FXR reporter mouse has basal luciferase expression in the terminal ileum, an organ with well-characterized FXRalpha signaling. In vivo luciferase activity reflected the diurnal activity pattern of the mouse, and is regulated by both natural (bile acids, chenodeoxycholic acid) and synthetic (GW4064) FXRalpha ligands. Moreover, in vivo and in vitro analysis showed luciferase activity after GW4064 administration in the liver, kidney, and adrenal gland, indicating that FXRalpha signaling is functional in these tissues. Hepatic luciferase activity was robustly induced in cholestatic mice, showing that FXRalpha signaling pathways are activated in this disease. In conclusion, we have developed an FXR reporter mouse that is useful to monitor FXRalpha signaling in vivo in health and disease. The use of this animal could facilitate the development of new therapeutic compounds that target FXRalpha in a tissue-specific manner.

  14. WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

    PubMed Central

    Liu, Qian; Chen, Haoqian; Ojode, Teresa; Gao, Xiangxi; Anaya-O'Brien, Sandra; Turner, Nicholas A.; Ulrick, Jean; DeCastro, Rosamma; Kelly, Corin; Cardones, Adela R.; Gold, Stuart H.; Hwang, Eugene I.; Wechsler, Daniel S.; Malech, Harry L.; Murphy, Philip M.

    2012-01-01

    WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4R334X, the most common truncation mutation in WHIM syndrome, CXCR4E343K mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4E343K had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling. PMID:22596258

  15. The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

    PubMed

    Schultze, B; Gross, H J; Brossmer, R; Herrler, G

    1991-11-01

    The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

  16. Inhibition of activation-induced apoptosis of thymocytes by all-trans- and 9-cis-retinoic acid is mediated via retinoic acid receptor alpha.

    PubMed

    Szondy, Z; Reichert, U; Bernardon, J M; Michel, S; Tóth, R; Karászi, E; Fésüs, L

    1998-05-01

    Thymocytes can be induced to undergo apoptotic cell death by activation through the T-cell receptor (TCR). This process requires macromolecular synthesis and has been shown to be inhibited by retinoic acids (RAs). Two groups of nuclear receptors for RAs have been identified: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). All-trans-RA is the high-affinity ligand for RARs, and 9-cis-RA additionally binds to RXRs with high affinity. Because 9-cis-RA is much more potent in inhibiting TCR-mediated death than all-trans-RA, it was suggested that RXRs participate in the process. In the present study various synthetic retinoid analogues were used to address this question further. The results presented suggest that the inhibitory effect of RAs on activation-induced death of thymocytes is mediated via RARalpha, because (1) it can be reproduced by various RARalpha analogues both in vitro and in vivo, (2) the effect of RAs can be inhibited by the addition of an RARalpha antagonist, (3) CD4+CD8+thymocytes, which die on TCR stimulation, express RARalpha. Stimulation of RARgamma, in contrast, enhances the activation-induced death of thymocytes and inhibits its prevention by RARalpha stimulation. RXR co-stimulation suspends this inhibitory effect of RARgamma and permits the preventive function of RARalpha on activation-induced death. Our results suggest a complex interaction between the various isoforms of retinoid receptors and demonstrate that low (physiological) concentrations of all-trans-RA do not affect the activation-induced death of thymocytes because the RARalpha-mediated inhibitory and the RARgamma-mediated enhancing pathways are in balance, whereas if 9-cis-RA is formed, additional stimulation of RXRs permits the inhibitory action of RARalpha.

  17. Inhibitory effects of nordihydroguaiaretic acid on ETA-receptor-mediated contractions to endothelin-1 in rat trachea.

    PubMed Central

    Henry, P. J.

    1994-01-01

    1. It has been shown previously that nordihydroguaiaretic acid (NDGA) inhibits endothelin-1 (ET-1)-induced contractions in rat isolated tracheal smooth muscle. To investigate the underlying mechanisms, this study examined the effects of NDGA on various aspects of the ETA and ETB receptor-effector systems which mediate ET-1-induced contractions in this preparation. 2. NDGA inhibited contractions induced by each of the isoforms of ET (ET-1, ET-2 and ET-3) but not those induced by the ETB receptor-selective agonist, sarafotoxin S6c, the cholinoceptor agonist, carbachol or the depolarizing spasmogen, KCl. 3. Quantitative autoradiographic studies of [125I]-ET-1 binding to rat tracheal smooth muscle indicated that NDGA was not an ET receptor antagonist. 4. NDGA inhibited the ETA receptor-mediated, intracellular Ca(2+)-dependent contractions induced by 100 nM ET-1 in Ca(2+)-free solution (by 75%, P < 0.01). Furthermore, NDGA markedly inhibited the contractions induced by ryanodine and cyclopiazonic acid; contractions purportedly due to Ca2+ release from intracellular stores. 5. Like NDGA, the sarcoplasmic reticulum Ca(2+)-ATPase inhibitors cyclopiazonic acid and thapsigargin inhibited contractions to ET-1, but not carbachol or KCl. However, cyclopiazonic acid, but not NDGA, also (a) induced transient contractions in rat trachea, (b) potentiated contractions induced by KCl, and (c) potentiated the extracellular Ca(2+)-dependent phase of ET-1-induced contractions, indicating that NDGA did not inhibit ET-1-induced contractions through Ca(2+)-ATPase inhibition and depletion of sarcoplasmic reticular Ca2+. 6. In control preparations, ET-1 induced a slowly developing, sustained contraction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8004399

  18. A study of the source-receptor relationships influencing the acidity of precipitation collected at a rural site in France

    NASA Astrophysics Data System (ADS)

    Charron, Aurélie; Plaisance, Hervé; Sauvage, Stéphane; Coddeville, Patrice; Galloo, Jean-Claude; Guillermo, René

    In order to examine the qualitative and quantitative source-receptor relationships responsible for acid rains at a background site in France, a receptor-oriented model was applied to the precipitation data collected from 1992 to 1995. Origins of acidic and alkaline species in precipitations have been investigated. The methodology combines precipitation chemical data with air parcel backward trajectories to establish concentration field maps of likely contributing sources. Highest acidities and concentrations of sulfate and nitrate in precipitation were associated with transport from the high emission areas of central Europe. Alkaline events were associated with air masses originating from Mediterranean basin or northern Africa. The quantitative relationships between the maps of potential sources and the European emissions of SO 2 and NO x were examined performing a correlation analysis. Good correlations were found between computed concentrations of acidic species and emissions of SO 2 and NO x. Substantial seasonal variations of acidic species were revealed. The highest concentrations occurred during the warm season. These seasonal variations are the effect of change of meteorological conditions and of the strength atmospheric processes according to the season.

  19. Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

    PubMed Central

    Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

    2015-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

  20. Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domain

    PubMed Central

    Ligneau, X; Morisset, S; Tardivel-Lacombe, J; Gbahou, F; Ganellin, C R; Stark, H; Schunack, W; Schwartz, J -C; Arrang, J -M

    2000-01-01

    Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)α-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species. PMID:11090094

  1. G protein coupled receptor signaled apoptosis is associated with activation of a cation insensitive acidic endonuclease and intracellular acidification.

    PubMed

    Sharma, K; Srikant, C B

    1998-01-01

    Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.

  2. C-terminal Domains of N-Methyl-d-aspartic Acid Receptor Modulate Unitary Channel Conductance and Gating*

    PubMed Central

    Maki, Bruce A.; Aman, Teresa K.; Amico-Ruvio, Stacy A.; Kussius, Cassandra L.; Popescu, Gabriela K.

    2012-01-01

    NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor. PMID:22948148

  3. Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland.

    PubMed

    Peiffer, A; Barden, N

    1987-06-01

    Using Northern blots and hybridization techniques, we have identified an approximately 6.5 kilobase glucocorticoid receptor mRNA species in rat anterior pituitary gland. Ovariectomy resulted in an approximately 2-fold increase in glucocorticoid receptor mRNA concentrations. This effect was maximal 8 days after surgery and glucocorticoid receptor mRNA levels remained elevated for at least up to 4 weeks. Administration of 17-beta-estradiol completely reversed the ovariectomy-induced increase in glucocorticoid receptor mRNA content of pituitary gland. Treatment of rats with corticosterone did not influence the ovariectomy-induced increase in glucocorticoid receptor mRNA content, indicating that this increase is not mediated via effects on circulating glucocorticoid levels or availability. In situ hybridization experiments confirmed the ovariectomy-induced increase in glucocorticoid receptor mRNA content and indicated that this action is widely distributed throughout the anterior pituitary gland.

  4. Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

    PubMed

    Dkhar, Hedwin Kitdorlang; Nanduri, Ravikanth; Mahajan, Sahil; Dave, Sandeep; Saini, Ankita; Somavarapu, Arun Kumar; Arora, Ashish; Parkesh, Raman; Thakur, Krishan Gopal; Mayilraj, Shanmugam; Gupta, Pawan

    2014-07-01

    The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

  5. Ligand-dependent activation of transcription in vitro by retinoic acid receptor α/retinoid X receptor α heterodimers that mimics transactivation by retinoids in vivo

    PubMed Central

    Dilworth, F. Jeffrey; Fromental-Ramain, Catherine; Remboutsika, Eumorphia; Benecke, Arndt; Chambon, Pierre

    1999-01-01

    All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARα and RXRα were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner. PMID:10051583

  6. Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

    PubMed

    Smith, Alison J; Oxley, Beth; Malpas, Sallie; Pillai, Gopalan V; Simpson, Peter B

    2004-11-01

    Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater

  7. Control of Gastric Acid Secretion in Somatostatin Receptor 2 Deficient Mice: Shift from Endocrine/Paracrine to Neurocrine Pathways

    PubMed Central

    Zhao, Chun-Mei; Martinez, Vicente; Piqueras, Laura; Wang, Lixin; Taché, Yvette; Chen, Duan

    2008-01-01

    The gastrin-enterochromaffin-like (ECL) cell-parietal cell axis is known to play an important role in the regulation of gastric acid secretion. Somatostatin, acting on somatostatin receptor type 2 (SSTR2), interferes with this axis by suppressing the activity of the gastrin cells, ECL cells, and parietal cells. Surprisingly, however, freely fed SSTR2 knockout mice seem to display normal circulating gastrin concentration and unchanged acid output. In the present study, we compared the control of acid secretion in these mutant mice with that in wild-type mice. In SSTR2 knockout mice, the number of gastrin cells was unchanged; whereas the numbers of somatostatin cells were reduced in the antrum (−55%) and increased in the oxyntic mucosa (35%). The ECL cells displayed a reduced expression of histidine decarboxylase and vesicle monoamine transport type 2 (determined by immunohistochemistry), and an impaired transformation of the granules to secretory vesicles (determined by electron microscopic analysis), suggesting low activity of the ECL cells. These changes were accompanied by an increased expression of galanin receptor type 1 in the oxyntic mucosa. The parietal cells were found to respond to pentagastrin or to vagal stimulation (evoked by pylorus ligation) with increased acid production. In conclusion, the inhibitory galanin-galanin receptor type 1 pathway is up-regulated in the ECL cells, and the direct stimulatory action of gastrin and vagal excitation is enhanced on the parietal cells in SSTR2 knockout mice. We suggest that there is a remodeling of the neuroendocrine mechanisms that regulate acid secretion in these mutant mice. PMID:17974627

  8. Expression of retinoic acid receptor genes in fetal and newborn rat lung.

    PubMed

    Grummer, M A; Thet, L A; Zachman, R D

    1994-04-01

    Lung differentiation and development are affected by vitamin A and its metabolites. One mechanism through which retinoids might exert their effects is through nuclear retinoic acid receptors (RAR). The gene expression profile of the RAR family (alpha, beta, gamma) has previously been determined in both the developing mouse embryo to 14.5 days gestation, and in the adult lung. The purpose of this study was to determine the expression of the RAR genes during the period of gestation that results in the formation of the saccular lung stage. Total RNA was extracted from fetal lungs of Sprague-Dawley rats at gestational days 17, 19, 20, 21, and 22, and from 12-hour-old newborns for Northern hybridization. Two transcripts of RAR alpha mRNA (3.7 and 2.7 kb) were found at each time point. At day 17, the 2.7 kb RAR alpha mRNA was increased two-fold or more than at any other time studied. At days 19-22 the levels of the 3.7 kb RAR alpha species were also lower than day 17 and newborn levels. One RAR beta mRNA transcript (3.4 kb), present at all time points, was significantly higher in the newborn than on days 17-22. Expression of RAR gamma mRNA could only be demonstrated by reverse transcriptase-polymerase chain reaction. We speculate that the higher RAR alpha species at day 17 indicates a role for RAR alpha in the maintenance of the columnar epithelial cells of the glandular phase of lung development.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8208594

  9. Sexually dimorphic adaptation of cardiac function: roles of epoxyeicosatrienoic acid and peroxisome proliferator-activated receptors.

    PubMed

    Qin, Jun; Le, Yicong; Froogh, Ghezal; Kandhi, Sharath; Jiang, Houli; Luo, Meng; Sun, Dong; Huang, An

    2016-06-01

    Epoxyeicosatrienoic acids (EETs) are cardioprotective mediators metabolized by soluble epoxide hydrolase (sEH) to form corresponding diols (DHETs). As a sex-susceptible target, sEH is involved in the sexually dimorphic regulation of cardiovascular function. Thus, we hypothesized that the female sex favors EET-mediated potentiation of cardiac function via downregulation of sEH expression, followed by upregulation of peroxisome proliferator-activated receptors (PPARs). Hearts were isolated from male (M) and female (F) wild-type (WT) and sEH-KO mice, and perfused with constant flow at different preloads. Basal coronary flow required to maintain the perfusion pressure at 100 mmHg was significantly greater in females than males, and sEH-KO than WT mice. All hearts displayed a dose-dependent decrease in coronary resistance and increase in cardiac contractility, represented as developed tension in response to increases in preload. These responses were also significantly greater in females than males, and sEH-KO than WT 14,15-EEZE abolished the sex-induced (F vs. M) and transgenic model-dependent (KO vs. WT) differences in the cardiac contractility, confirming an EET-driven response. Compared with M-WT controls, F-WT hearts expressed downregulation of sEH, associated with increased EETs and reduced DHETs, a pattern comparable to that observed in sEH-KO hearts. Coincidentally, F-WT and sEH-KO hearts exhibited increased PPARα expression, but comparable expression of eNOS, PPARβ, and EET synthases. In conclusion, female-specific downregulation of sEH initiates an EET-dependent adaptation of cardiac function, characterized by increased coronary flow via reduction in vascular resistance, and promotion of cardiac contractility, a response that could be further intensified by PPARα.

  10. Pharmacologic retinoid signaling and physiologic retinoic acid receptor signaling inhibit basal cell carcinoma tumorigenesis.

    PubMed

    So, Po-Lin; Fujimoto, Michele A; Epstein, Ervin H

    2008-05-01

    Basal cell carcinoma (BCC) is the most common human cancer. Patients with basal cell nevus syndrome (Gorlin syndrome) are highly susceptible to developing many BCCs as a result of a constitutive inactivating mutation in one allele of PATCHED 1, which encodes a tumor suppressor that is a major inhibitor of Hedgehog signaling. Dysregulated Hedgehog signaling is a common feature of both hereditary and sporadic BCCs. Recently, we showed remarkable anti-BCC chemopreventive efficacy of tazarotene, a retinoid with retinoic acid receptor (RAR) beta/gamma specificity, in Ptch1+/- mice when treatment was commenced before carcinogenic insults. In this study, we assessed whether the effect of tazarotene against BCC carcinogenesis is sustained after its withdrawal and whether tazarotene is effective against preexisting microscopic BCC lesions. We found that BCCs did not reappear for at least 5 months after topical drug treatment was stopped and that already developed, microscopic BCCs were susceptible to tazarotene inhibition. In vitro, tazarotene inhibited a murine BCC keratinocyte cell line, ASZ001, suggesting that its effect in vivo is by direct action on the actual tumor cells. Down-regulation of Gli1, a target gene of Hedgehog signaling and up-regulation of CRABPII, a target gene of retinoid signaling, were observed with tazarotene treatment. Finally, we investigated the effects of topical applications of other retinoid-related compounds on BCC tumorigenesis in vivo. Tazarotene was the most effective of the preparations studied, and its effect most likely was mediated by RARgamma activation. Furthermore, inhibition of basal RAR signaling in the skin promoted BCC carcinogenesis, suggesting that endogenous RAR signaling restrains BCC growth.

  11. ACTIVATION OF MOUSE AND HUMAN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPAR ALPHA, GAMMA, BETA DELTA) BY PERFLUOROOCTANOIC ACID (PFOA) AND PERFLUOROOCTANE SULFONATE (PFOS)

    EPA Science Inventory

    This study evaluates the potential for perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) to activate peroxisome proliferator-activated receptors (PPARs), using a transient transfection cell assay. Cos-1 cells were cultured in DMEM with fetal bovine serum (FBS) in ...

  12. Associations of Uric Acid with Polymorphisms in the δ-Aminolevulinic Acid Dehydratase, Vitamin D Receptor, and Nitric Oxide Synthase Genes in Korean Lead Workers

    PubMed Central

    Weaver, Virginia M.; Schwartz, Brian S.; Jaar, Bernard G.; Ahn, Kyu-Dong; Todd, Andrew C.; Lee, Sung-Soo; Kelsey, Karl T.; Silbergeld, Ellen K.; Lustberg, Mark E.; Parsons, Patrick J.; Wen, Jiayu; Lee, Byung-Kook

    2005-01-01

    Recent research suggests that uric acid may be nephrotoxic at lower levels than previously recognized and that it may be one mechanism for lead-related nephrotoxicity. Therefore, in understanding mechanisms for lead-related nephrotoxicity, it would be of value to determine whether genetic polymorphisms that are associated with renal outcomes in lead workers and/or modify associations between lead dose and renal function are also associated with uric acid and/or modify associations between lead dose and uric acid. We analyzed data on three such genetic polymorphisms: δ-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR). Mean (± SD) tibia, blood, and dimercaptosuccinic acid–chelatable lead levels were 37.2 ± 40.4 μg/g bone mineral, 32.0± 15.0 g/dL, and 0.77± 0.86 μg/mg creatinine, respectively, in 798 current and former lead workers. Participants with the eNOS Asp allele had lower mean serum uric acid compared with those with the Glu/Glu genotype. Among older workers (age ≥ median of 40.6 years), ALAD genotype modified associations between lead dose and uric acid levels. Higher lead dose was significantly associated with higher uric acid in workers with the ALAD1-1 genotype; associations were in the opposite direction in participants with the variant ALAD1-2 genotype. In contrast, higher tibia lead was associated with higher uric acid in those with the variant VDR B allele; however, modification was dependent on participants with the bb genotype and high tibia lead levels. We conclude that genetic polymorphisms may modify uric acid mediation of lead-related adverse renal effects. PMID:16263504

  13. QUANTITATION OF ABERRANT INTERLOCUS T-CELL RECEPTOR REARRANGEMENTS IN MOUSE THYMOCYTES AND THE EFFECT OF THE HERBICIDE 2,4- DICHLOROPHENOXYACETIC ACID

    EPA Science Inventory

    Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4- Dichlorophenoxyacetic acid

    Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene a...

  14. Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

    SciTech Connect

    Wu, Weibin; Liu, Xijun; Peng, Xiaomin; Xue, Ruyi; Ji, Lingling; Shen, Xizhong; Chen, She; Gu, Jianxin; Zhang, Si

    2014-05-23

    Highlights: • FXR deficiency enhanced MCD diet-induced hepatic fibrosis. • FXR deficiency attenuated MCD diet-induced hepatic steatosis. • FXR deficiency repressed genes involved in fatty acid uptake and triglyceride accumulation. - Abstract: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR{sup −/−}) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR{sup −/−} mice fed MCD diet (FXR{sup −/−}/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR{sup −/−}/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR{sup −/−}/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR{sup −/−}/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.

  15. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    SciTech Connect

    Okito, Asuka; Nakahama, Ken-ichi; Akiyama, Masako; Ono, Takashi; Morita, Ikuo

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  16. Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma.

    PubMed

    Arici, Mustafa; Chana, Ravinder; Lewington, Andrew; Brown, Jez; Brunskill, Nigel John

    2003-01-01

    In nephrotic syndrome, large quantities of albumin enter the kidney tubule. This albumin carries with it a heavy load of fatty acids to which the proximal tubule cells are exposed at high concentration. It is postulated that exposure to fatty acids in this way is injurious to proximal tubule cells. This study has examined the ability of fatty acids to interact with peroxisome proliferator-activated receptors (PPAR) in primary cultures of human proximal tubule cells. Luciferase reporter assays in transiently transfected human proximal tubule cells were used to show that albumin bound fatty acids and other agonists activate PPARgamma in a dose-dependent manner. One of the consequences of this activation is apoptosis of the cells as determined by changes in cell morphology, evidence of PARP cleavage, and appearance of DNA laddering. Overexpression of PPARgamma in these cells also results in enhanced apoptosis. Both fatty acid-induced PPAR activation and apoptosis in these cells can be blocked by PPAR response element decoy oligonucleotides. Activation of PPARgamma by the specific agonist PGJ(2) is associated with inhibition of cell proliferation, whereas activation by albumin bound fatty acids is accompanied by increased proliferation. However, the net balance of apoptosis/proliferation favors deletion of cells. These results implicate albumin-bound fatty acids as important mediators of tubular injury in nephrosis and provide fresh impetus for pursuit of lipid-lowering strategies in proteinuric renal disease. PMID:12506134

  17. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    SciTech Connect

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.; Lurz, Rudi; Calendar, Richard; Klumpp, Jochen; Loessner, Martin J.

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  18. Steady state critical loads of acidity for sulphur and nitrogen: a multi-receptor, multi-criterion approach.

    PubMed

    Aherne, J; Farrell, E P

    2002-04-15

    The critical load approach to emission reductions has become an important element of the United Nations Economic Commission for Europe (UNECE) Convention on Long-Range Transboundary Air Pollution (CLRTAP). The steady state mass balance approach (SSMB) is the most widely used method in Europe for estimating critical loads, typically applied to forest ecosystems on mineral soils in conjunction with a base cation to aluminium (Bc:Al) ratio using a default critical limit of 1.0. The 'typical' approach has been expanded in this paper by: inclusion of a broader range of natural and semi-natural receptor ecosystems; inclusion of multiple chemical criterion for each receptor; and an attempt to include organic soils (peatlands). Critical loads of acidity were estimated for the Republic of Ireland using four receptor ecosystems (coniferous forest, deciduous forest, natural grasslands and moors and heathlands) and seven chemical criteria. The dominant chemical criteria, in the determination of critical loads, were based on a critical pH limit for mineral soils or a critical pH shift in relation to pristine conditions for organic soils. Approximately 68 and 26% of the final distribution of critical loads were estimated using these criteria, respectively. The 5th-percentile critical loads of acidity were more sensitive than those previously estimated for Ireland due to the inclusion of organic soils. Furthermore, coniferous ecosystems had the lowest critical loads due to the high percentage occurring on organic soils, and the removal of base cations through harvesting. The results demonstrate that it may be more appropriate to use multiple criteria and receptors to ensure adequate protection of biological indicators. However, it is important that appropriate critical limits are chosen to protect the biological indicators and receptor ecosystems from long-term damage.

  19. Excitatory Amino Acid Receptors Mediate Asymmetry and Lateralization in the Descending Cardiovascular Pathways from the Dorsomedial Hypothalamus

    PubMed Central

    Xavier, Carlos Henrique; Ianzer, Danielle; Lima, Augusto Martins; Marins, Fernanda Ribeiro; Pedrino, Gustavo Rodrigues; Vaz, Gisele; Menezes, Gustavo Batista; Nalivaiko, Eugene; Fontes, Marco Antônio Peliky

    2014-01-01

    The dorsomedial hypothalamus (DMH) and lateral/dorsolateral periaqueductal gray (PAG) are anatomically and functionally connected. Both the DMH and PAG depend on glutamatergic inputs for activation. We recently reported that removal of GABA-ergic tone in the unilateral DMH produces: asymmetry, that is, a right- (R-) sided predominance in cardiac chronotropism, and lateralization, that is, a greater increase in ipsilateral renal sympathetic activity (RSNA). In the current study, we investigated whether excitatory amino acid (EAA) receptors in the DMH–PAG pathway contribute to the functional interhemispheric difference. In urethane (1.2 to 1.4 g/kg, i.p.) anesthetized rats, we observed that: (i) nanoinjections of N-methyl D-aspartate (NMDA 100 pmol/100 nl) into the unilateral DMH produced the same right-sided predominance in the control of cardiac chronotropy, (ii) nanoinjections of NMDA into the ipsilateral DMH or PAG evoked lateralized RSNA responses, and (iii) blockade of EAA receptors in the unilateral DMH attenuated the cardiovascular responses evoked by injection of NMDA into either the R- or left- (L-) PAG. In awake rats, nanoinjection of kynurenic acid (1 nmol/100 nL) into the L-DMH or R- or L-PAG attenuated the tachycardia evoked by air stress. However, the magnitude of stress-evoked tachycardia was smallest when the EAA receptors of the R-DMH were blocked. We conclude that EAA receptors contribute to the right-sided predominance in cardiac chronotropism. This interhemispheric difference that involves EAA receptors was observed in the DMH but not in the PAG. PMID:25397884

  20. Design and synthesis of conformationally constrained 3-(N-alkylamino)propylphosphonic acids as potent agonists of sphingosine-1-phosphate (S1P) receptors.

    PubMed

    Yan, Lin; Hale, Jeffrey J; Lynch, Christopher L; Budhu, Richard; Gentry, Amy; Mills, Sander G; Hajdu, Richard; Keohane, Carol Ann; Rosenbach, Mark J; Milligan, James A; Shei, Gan-Ju; Chrebet, Gary; Bergstrom, James; Card, Deborah; Rosen, Hugh; Mandala, Suzanne M

    2004-10-01

    A series of conformationally constrained 3-(N-alkylamino)propylphosphonic acids were systematically synthesized and their activities as S1P receptor agonists were evaluated. Several pyrrolidine and cyclohexane analogs had S1P receptor profiles comparable to the acyclic lead compound, 3-(N-tetradecylamino)propylphosphonic acid (3), lowered circulating lymphocytes in mice after iv administration and were thus identified as being suitable for further investigations.

  1. Low density lipoprotein receptor related protein 1 variant interacts with saturated fatty acids in Puerto Ricans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low density lipoprotein related receptor protein 1 (LRP1) is a multi-functional endocytic receptor that is highly expressed in adipocytes and the hypothalamus. Animal models and in vitro studies support a role for LRP1 in adipocyte metabolism and leptin signaling, but genetic polymorphisms have not ...

  2. Identification of miR-26a as a Target Gene of Bile Acid Receptor GPBAR-1/TGR5

    PubMed Central

    Chen, Xiaosong; Xu, Haixia; Ding, Lili; Lou, Guiyu; Liu, Yan; Yao, Yalan; Chen, Liangwan; Huang, Wendong; Fu, Xianghui

    2015-01-01

    GPBAR1/TGR5 is a G protein–coupled receptor of bile acids. TGR5 is known to regulate the BA homeostasis and energy metabolism. Recent studies highlight an important role of TGR5 in alleviating obesity and improving glucose regulation, however, the mechanism of which is still unclear. Here we report that TGR5 is involved in mediating the anti-obesity and anti-hyperglycemia effect of a natural compound, oleanolic acid. By comparing the miRNA profiles between wild type and TGR5-/- livers after OA treatment, we identified miR-26a as a novel downstream target gene of TGR5 activation. The expression of miR-26a in the liver was induced in a TGR5-dependent manner after feeding the mice with a bile acid diet. TGR5 activation strongly increased the expression of miR-26a in macrophages, including the Kupffer cells in the liver. We further demonstrated that JNK pathway was required for miR-26a induction by TGR5 activation. Interestingly, we located the TGR5-responsive DNA element to a proximal region of miR-26’s promoter, which was independent of the transcription of its host genes. These results unravel a new mechanism by which bile acid receptor TGR5 activates a miRNA gene expression. PMID:26107166

  3. Glucuronic acid and the ethanol metabolite ethyl-glucuronide cause Toll-like receptor 4 activation and enhanced pain

    PubMed Central

    Lewis, Susannah S.; Hutchinson, Mark R.; Zhang, Yingning; Hund, Dana K.; Maier, Steven F.; Rice, Kenner C.; Watkins, Linda R.

    2013-01-01

    We have previously observed that the non-opioid morphine metabolite, morphine-3-glucuronide, enhances pain via a toll-like receptor 4 (TLR4) dependent mechanism. The present studies were undertaken to determine whether TLR4-dependent pain enhancement generalizes to other classes of glucuronide metabolites. In silico modeling predicted that glucuronic acid alone and ethyl glucuronide, a minor but long-lasting ethanol metabolite, would dock to the same MD-2 portion of the TLR4 receptor complex previously characterized as the docking site for morphine-3-glucuronide. Glucuronic acid, ethyl glucuronide and ethanol all caused an increase in TLR4-dependent reporter protein expression in a cell line transfected with TLR4 and associated co-signaling molecules. Glucuronic acid-, ethyl glucuronide-, and ethanol-induced increases in TLR4 signaling were blocked by the TLR4 antagonists LPS-RS and (+)-naloxone. Glucuronic acid and ethyl glucuronide both caused allodynia following intrathecal injection in rats, which was blocked by intrathecal co-administration of the TLR4 antagonist LPS-RS. The finding that ethyl glucuronide can cause TLR4-dependent pain could have implications for human conditions such as hangover headache and alcohol withdrawal hyperalgesia, as well as suggesting that other classes of glucuronide metabolites could have similar effects. PMID:23348028

  4. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    PubMed Central

    Lo, Shih-Hsiang; Cheng, Kai-Chung; Li, Ying-Xiao; Chang, Chin-Hong; Cheng, Juei-Tang; Lee, Kung-Shing

    2016-01-01

    Background G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time. Conclusion Similar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future. PMID:27578964

  5. Galpha12/13- and rho-dependent activation of phospholipase C-epsilon by lysophosphatidic acid and thrombin receptors.

    PubMed

    Hains, Melinda D; Wing, Michele R; Maddileti, Savitri; Siderovski, David P; Harden, T Kendall

    2006-06-01

    Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via

  6. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  7. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  8. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

    PubMed

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-04-12

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  9. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    PubMed Central

    Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  10. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons

    PubMed Central

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (IGABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited IGABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation. PMID:27616980

  11. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons

    PubMed Central

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (IGABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited IGABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation.

  12. Adolescent social defeat alters N-methyl-D-aspartic acid receptor expression and impairs fear learning in adulthood.

    PubMed

    Novick, Andrew M; Mears, Mackenzie; Forster, Gina L; Lei, Yanlin; Tejani-Butt, Shanaz M; Watt, Michael J

    2016-05-01

    Repeated social defeat of adolescent male rats results in adult mesocortical dopamine hypofunction, impaired working memory, and increased contextual anxiety-like behavior. Given the role of glutamate in dopamine regulation, cognition, and fear and anxiety, we investigated potential changes to N-methyl-D-aspartic acid (NMDA) receptors following adolescent social defeat. As both NMDA receptors and mesocortical dopamine are implicated in the expression and extinction of conditioned fear, a separate cohort of rats was challenged with a classical fear conditioning paradigm to investigate whether fear learning is altered by adolescent defeat. Quantitative autoradiography was used to measure 3H-MK-801 binding to NMDA receptors in regions of the medial prefrontal cortex, caudate putamen, nucleus accumbens, amygdala and hippocampus. Assessment of fear learning was achieved using an auditory fear conditioning paradigm, with freezing toward the auditory tone used as a measure of conditioned fear. Compared to controls, adolescent social defeat decreased adult NMDA receptor expression in the infralimbic region of the prefrontal cortex and central amygdala, while increasing expression in the CA3 region of the hippocampus. Previously defeated rats also displayed decreased conditioned freezing during the recall and first extinction periods, which may be related to the observed decreases and increases in NMDA receptors within the central amygdala and CA3, respectively. The alteration in NMDA receptors seen following adolescent social defeat suggests that dysfunction of glutamatergic systems, combined with mesocortical dopamine deficits, likely plays a role in the some of the long-term behavioral consequences of social stressors in adolescence seen in both preclinical and clinical studies. PMID:26876136

  13. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons.

    PubMed

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (I GABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited I GABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of I GABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation. PMID:27616980

  14. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.

    PubMed

    Minkoff, Benjamin B; Stecker, Kelly E; Sussman, Michael R

    2015-05-01

    Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  15. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors

    PubMed Central

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-01-01

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic a