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Sample records for p2 receptor-mediated signaling

  1. P2y receptor-mediated angiogenesis via vascular endothelial growth factor receptor 2 signaling.

    PubMed

    Rumjahn, Sharif M; Baldwin, Karla A; Buxton, Iain L O

    2007-01-01

    Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 microM ATP and 10 microM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 microM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis.

  2. P2y Receptor-Mediated Angiogenesis via Vascular Endothelial Growth Factor Receptor 2 Signaling

    PubMed Central

    Rumjahn, Sharif M.; Baldwin, Karla A; Buxton, Iain L. O.

    2011-01-01

    Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 μM ATP and 10 μM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 μM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis. PMID:18605230

  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. Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia

    PubMed Central

    Nagy, Béla; Jin, Jianguo; Ashby, Barrie; Reilly, Michael P.; Kunapuli, Satya P.

    2011-01-01

    Summary Background In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective This study was carried out to investigate the contribution of the ADP receptor P2Y12-mediated pathway in platelet hyperreactivity due to hypercholesterolemia. Methods Low-density lipoprotein receptor deficient mice and C57Bl/6 wild type mice were fed on normal chow and high-fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation, secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal-regulated protein kinase (ERK) and Akt were analyzed. Results Plasma cholesterol levels ranged from 69±10 to 1011±185 mg/dl depending on diet in mice with different genotypes. Agonist-dependent aggregation, dense and α-granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels even if elevated thromboxane generation was blocked. These functional responses were induced via increased level of Gi mediated ERK and Akt phosphorylation in hypercholesterolemic mice versus normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR-C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared to normocholesterolemic controls. Conclusions These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia. PMID:21261805

  5. P2X2 and P2X5 Receptors Mediate Bladder Hyperesthesia in ICC in Female Overactive Bladder.

    PubMed

    Meng, Mingsen; Zheng, Ji; Yan, Junan; Li, Qianwei; Fang, Qiang; Li, Weibing

    2015-06-01

    This study was set to explore the role of P2X2 and P2X5 as the important molecules in sensory afferent of bladder in female overactive bladder (OAB) patients with the bladder hyperesthesia. Sixty-eight OAB patients admitted in Southwest Hospital affiliated to the Third Military Medical University during September, 2011-December, 2012 were selected and included in the experimental group (OAB group) and 30 healthy volunteers during the same period were included as the control group. We recorded voiding diary and urodynamic results, and immunohistochemistry analysis was used to detect P2X2 and P2X5 receptor in interstitial cell of Caja (ICC) in bladder tissue of female OAB patients and healthy volunteers, to tentatively explore the effect of P2X2 and P2X5 in bladder hyperesthesia. Urodynamic study has important diagnostic value in the diagnosis and differential diagnosis of OAB. P2X2 receptor was significantly up-regulated in bladder ICC in OAB group. The blockage of P2X2 receptor could significantly inhibit the contraction of bladder muscle strips, decrease the bladder pressure and the electric discharge of pelvic nerve. PET and urodynamic study showed that micturition desire sense in PAG area of pons in OAB patients was significantly increased compared with the control group. The up-regulation of P2X2 in ICC is an important factor to cause bladder hyperesthesia in OAB patients. PET and urodynamic study indicate that the bladder-originated nervous impulses are important cause of OAB. This study provides a basis for the study of P2X2 receptor in ICC in bladder hyperesthesia of OAB patients.

  6. P2U-receptor mediated endothelium-dependent but nitric oxide-independent vascular relaxation

    PubMed Central

    Malmsjö, M; Edvinsson, L; Erlinge, D

    1998-01-01

    −3 M) totally abolished the remaining ATP and UTP-dilatation. This indicates a dilatation mediated by an endothelium-dependent non-NO factor, probably EDHF. Agonist potency (UTP>ATP≈#62;2-MeSATP), indicates that the EDHF-mediated dilatation was stimulated by a P2U-receptor, possibly by a selective pyrimidine-receptor. In contrast, a P2Y-receptor stimulated NO-mediated dilatation (2-MeSATP=ATP>UTP). In conclusion, the dilator effects of ATP and especially UTP can be mediated by an endothelium-dependent non-NO-mediated mechanism, probably EDHF, mediated by a P2U-receptor, possibly a selective pyrimidine-receptor, while NO-mediated dilatation is stimulated mainly by a P2Y1-receptor. Furthermore, the EDHF-dilatation is dependent on the resting tone of the blood vessel. PMID:9517392

  7. P2U-receptor mediated endothelium-dependent but nitric oxide-independent vascular relaxation.

    PubMed

    Malmsjö, M; Edvinsson, L; Erlinge, D

    1998-02-01

    (-3) M) totally abolished the remaining ATP and UTP-dilatation. This indicates a dilatation mediated by an endothelium-dependent non-NO factor, probably EDHF. 5. Agonist potency (UTP>ATP>2-MeSATP), indicates that the EDHF-mediated dilatation was stimulated by a P2U-receptor, possibly by a selective pyrimidine-receptor. In contrast, a P2Y-receptor stimulated NO-mediated dilatation (2-MeSATP=ATP>UTP). 6. In conclusion, the dilator effects of ATP and especially UTP can be mediated by an endothelium-dependent non-NO-mediated mechanism, probably EDHF, mediated by a P2U-receptor, possibly a selective pyrimidine-receptor, while NO-mediated dilatation is stimulated mainly by a P2Y1-receptor. Furthermore, the EDHF-dilatation is dependent on the resting tone of the blood vessel.

  8. P2X7 receptor-mediated calcium dynamics in HEK293 cells: experimental characterization and modelling approach

    NASA Astrophysics Data System (ADS)

    Di Garbo, A.; Alloisio, S.; Nobile, M.

    2012-04-01

    The P2X7 receptor (P2X7R) induces ionotropic Ca2 + signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca2 + variations evoked by 3‧-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca2 + dynamics in HEK293. Our model gives an account of the ionotropic Ca2 + influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca2 + responses evoked by BzATP, the model predicted that an impairment in Ca2 + extrusion flux through the plasma membrane is a key factor for Ca2 + homeostasis in HEK293 cells.

  9. P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro.

    PubMed

    Alvares, T S; Revill, A L; Huxtable, A G; Lorenz, C D; Funk, G D

    2014-07-15

    PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y1R) activation. Despite this, and the fact that inspiratory MNs express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mm) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca(2+)-dependent non-selective cation current ICAN; high intracellular concentrations of BAPTA, which buffers Ca(2+) increases necessary for activation of ICAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs. PMID:24879869

  10. P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

    PubMed Central

    Kim, Ji Yang; Ko, Ah-Reum; Kim, Ji-Eun

    2015-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE) in the distinct brain regions. In addition, P2X7 receptor (P2X7R), an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death). Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths. PMID:26388738

  11. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  12. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  13. P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages1

    PubMed Central

    Lees, Michael P.; Fuller, Stephen J.; McLeod, Rima; Boulter, Nicola R.; Miller, Catherine M.; Zakrzewski, Alana M.; Mui, Ernest J.; Witola, William H.; Coyne, Jessica J.; Hargrave, Aubrey C.; Jamieson, Sarra E.; Blackwell, Jenefer M.; Wiley, James S.; Smith, Nicholas C.

    2010-01-01

    The P2X7 receptor (P2X7R)4 is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms (SNPs) that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this paper we show that macrophages from people with the 1513C (rs3751143) loss-of-function P2X7R SNP are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knock-out mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production. PMID:20488797

  14. P2X3, but not P2X1, receptors mediate ATP-activated current in neurons innervating tooth-pulp.

    PubMed

    Liu, Yu-wei; Chen, Xiao-qing; Tian, Xiang; Chen, Lin; Wu, Yu-xiang; Huang, Dan; Yi, Hui-ling; Yi, Chu-li; Li, Chao-ying

    2013-06-01

    We developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons and to conduct single cell immunohistochemical staining for P2X1 and P2X3 subunits in the same neuron. Three types of ATP-activated current in these neurons (F, I and S) were recorded. The cells exhibiting the type F current mainly showed positive staining for P2X3, but negative staining for P2X1. The results provide direct and convincing evidence at the level of single native nociceptive neurons for correlation of the characteristics of ATP-activated currents with their composition of P2X1 and P2X3 subunits and cell size. The results also suggest that the P2X3, but not P2X1, is the main subunit that mediates the fast ATP-activated current in nociceptive neurons.

  15. Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

    PubMed

    Liu, Bo; Zheng, Yonggang; Yin, Feng; Yu, Jianzhong; Silverman, Neal; Pan, Duojia

    2016-01-28

    The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings. PMID:26824654

  16. Aripiprazole has functionally selective actions at dopamine D2 receptor-mediated signaling pathways.

    PubMed

    Urban, Jonathan D; Vargas, Gabriel A; von Zastrow, Mark; Mailman, Richard B

    2007-01-01

    Aripiprazole is a unique atypical antipsychotic drug with an excellent side-effect profile presumed, in part, to be due to lack of typical D(2) dopamine receptor antagonist properties. Whether aripiprazole is a typical D(2) partial agonist, or a functionally selective D(2) ligand, remains controversial (eg D(2)-mediated inhibition of adenylate cyclase is system dependent; aripiprazole antagonizes D(2) receptor-mediated G-protein-coupled inwardly rectifying potassium channels and guanosine triphosphate nucleotide (GTP)gammaS coupling). The current study examined the D(2L) receptor binding properties of aripiprazole, as well as the effects of the drug on three downstream D(2) receptor-mediated functional effectors: mitogen-activated protein kinase (MAPK) phosphorylation, potentiation of arachidonic acid (AA) release, and D(2) receptor internalization. Unlike quinpirole (a full D(2) agonist) or (-)3PPP (S(-)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride, a D(2) partial agonist), the apparent D(2) affinity of aripiprazole was not decreased significantly by GTP. Moreover, full or partial agonists are expected to have Hill slopes <1.0, yet that of aripiprazole was significantly >1.0. Whereas aripiprazole partially activated both the MAPK and AA pathways, its potency vs MAPK phosphorylation was much lower relative to potencies in assays either of AA release or inhibition of cyclic adenosine 3',5'-cyclic monophosphate accumulation. In addition, unlike typical agonists, neither aripiprazole nor (-)3PPP produced significant internalization of the D(2L) receptor. These data are clear evidence that aripiprazole affects D(2L)-mediated signaling pathways in a differential manner. The results are consistent with the hypothesis that aripiprazole is a functionally selective D(2) ligand rather than a simple partial agonist. Such data may be useful in understanding the novel clinical actions of this drug.

  17. Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

    PubMed

    Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B

    2014-04-30

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

  18. P2X and P2Y receptor signaling in red blood cells

    PubMed Central

    Sluyter, Ronald

    2015-01-01

    Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation, and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology. PMID:26579528

  19. Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells.

    PubMed

    Raqeeb, Abdul; Sheng, Jianzhong; Ao, Ni; Braun, Andrew P

    2011-04-01

    In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.

  20. The P2Y2 nucleotide receptor mediates UTP-induced vascular cell adhesion molecule-1 expression in coronary artery endothelial cells.

    PubMed

    Seye, Cheikh I; Yu, Ningpu; Jain, Renu; Kong, Qiongman; Minor, Tess; Newton, Jessica; Erb, Laurie; González, Fernando A; Weisman, Gary A

    2003-07-01

    P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1-100 microm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-alpha. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis.

  1. P2Y2 Receptor-mediated Lymphotoxin-α Secretion Regulates Intercellular Cell Adhesion Molecule-1 Expression in Vascular Smooth Muscle Cells*

    PubMed Central

    Seye, Cheikh I.; Agca, Yuksel; Agca, Cansu; Derbigny, Wilbert

    2012-01-01

    The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y2 nucleotide receptor (P2Y2R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y2R−/− SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y2R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y2R deficient in LTA secretion. These data suggest that P2Y2R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells. PMID:22298782

  2. Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Sawada, C; Higuchi, H; Teraoka, H; Yamaguchi, M

    1997-01-01

    Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces. PMID:9343828

  3. The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells

    PubMed Central

    Dissmore, Tixieanna; Seye, Cheikh I.; Medeiros, Denis M.; Weisman, Gary A.; Bardford, Barry; Mamedova, Laman

    2016-01-01

    Background and aims The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. Methods Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R−/− mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). Results P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R−/− VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R−/− VSMCs versus cells transfected with the mutant P2Y2R. Conclusions Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A. PMID:27522265

  4. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  5. The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1).

    PubMed

    Seye, Cheikh I; Yu, Ningpu; González, Fernando A; Erb, Laurie; Weisman, Gary A

    2004-08-20

    UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.

  6. Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

    PubMed

    Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

    2015-10-01

    Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses.

  7. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    SciTech Connect

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  8. α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice.

    PubMed

    Ding, Fengfei; O'Donnell, John; Thrane, Alexander S; Zeppenfeld, Douglas; Kang, Hongyi; Xie, Lulu; Wang, Fushun; Nedergaard, Maiken

    2013-12-01

    Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.

  9. Inhibition of PAR-4 and P2Y12 receptor-mediated platelet activation produces distinct hepatic pathologies in experimental xenobiotic-induced cholestatic liver disease.

    PubMed

    Joshi, Nikita; Kopec, Anna K; Ray, Jessica L; Luyendyk, James P

    2016-07-15

    Emerging evidence supports a protective effect of platelets in experimental cholestatic liver injury and cholangiofibrosis. Coagulation-mediated platelet activation has been shown to inhibit experimental chronic cholestatic liver necrosis and biliary fibrosis. This occurs through thrombin-mediated activation of protease activated receptor-4 (PAR-4) in mice. However, it is not known whether other pathways of platelet activation, such as adenosine diphosphate (ADP)-mediated receptor P2Y12 activation is also protective. We tested the hypothesis that inhibition of P2Y12-mediated platelet activation exacerbates hepatic injury and cholangiofibrosis, and examined the impact of P2Y12 inhibition in both the presence and absence of PAR-4. Treatment of wild-type mice with the P2Y12 receptor antagonist clopidogrel increased biliary hyperplasia and cholangiofibrosis in wild-type mice exposed to the xenobiotic alpha-naphthylisothiocyanate (ANIT) for 4 weeks compared to vehicle-treated mice exposed to ANIT. Interestingly, this effect of clopidogrel occurred without a corresponding increase in hepatocellular necrosis. Whereas biliary hyperplasia and cholangiofibrosis were increased in PAR-4(-/-) mice, clopidogrel treatment failed to further increase these pathologies in PAR-4(-/-) mice. The results indicate that inhibition of receptor P2Y12-mediated platelet activation exacerbates bile duct fibrosis in ANIT-exposed mice, independent of hepatocellular necrosis. Moreover, the lack of an added effect of clopidogrel administration on the exaggerated pathology in ANIT-exposed PAR-4(-/-) mice reinforces the prevailing importance of coagulation-mediated platelet activation in limiting this unique liver pathology. PMID:27475285

  10. Essential role of endocytosis for interleukin-4-receptor-mediated JAK/STAT signalling.

    PubMed

    Kurgonaite, Kristina; Gandhi, Hetvi; Kurth, Thomas; Pautot, Sophie; Schwille, Petra; Weidemann, Thomas; Bökel, Christian

    2015-10-15

    Many important signalling cascades operate through specialized signalling endosomes, but a corresponding mechanism has as yet not been described for hematopoietic cytokine receptors. Based on live-cell affinity measurements, we recently proposed that ligand-induced interleukin-4 receptor (IL-4R) complex formation and thus JAK/STAT pathway activation requires a local subcellular increase in receptor density. Here, we show that this concentration step is provided by the internalization of IL-4R subunits through a constitutive, Rac1-, Pak- and actin-mediated endocytosis route that causes IL-4R subunits to become enriched by about two orders of magnitude within a population of cortical endosomes. Consistently, ligand-induced receptor dimers are preferentially detected within these endosomes. IL-4 signalling can be blocked by pharmacological inhibitors targeting the actin polymerization machinery driving receptor internalization, placing endocytosis unambigously upstream of receptor activation. Taken together, these observations demonstrate a role for endocytosis that is mechanistically distinct from the scaffolding function of signalling endosomes in other pathways.

  11. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress.

    PubMed

    Evanson, Nathan K; Herman, James P

    2015-10-15

    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  12. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    PubMed

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  13. Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication

    PubMed Central

    Liang, Yong; Yang, Kaiting; Guo, Jingya; Wroblewska, Joanna; Fu, Yang-Xin; Peng, Hua

    2015-01-01

    Host anti-viral innate immunity plays important roles in the defense against HSV-1 infection. In this study, we find an unexpected role for innate LT/LIGHT signaling in promoting HSV-1 replication and virus induced inflammation in immunocompromised mice. Using a model of footpad HSV-1 infection in Rag1–/– mice, we observed that blocking LT/LIGHT signaling with LTβR-Ig could significantly delay disease progression and extend the survival of infected mice. LTβR-Ig treatment reduced late proinflammatory cytokine release in the serum and nervous tissue, and inhibited chemokine expression and inflammatory cells infiltration in the dorsal root ganglia (DRG). Intriguingly, LTβR-Ig treatment restricted HSV-1 replication in the DRG but not the footpad. These findings demonstrate a critical role for LT/LIGHT signaling in modulating innate inflammation and promoting HSV-1 replication in the nervous system, and suggest a new target for treatment of virus-induced adverse immune response and control of severe HSV-1 infection. PMID:25993659

  14. 3-iodothyronamine differentially modulates α-2A-adrenergic receptor-mediated signaling.

    PubMed

    Dinter, Juliane; Mühlhaus, Jessica; Jacobi, Simon Friedrich; Wienchol, Carolin Leonie; Cöster, Maxi; Meister, Jaroslawna; Hoefig, Carolin Stephanie; Müller, Anne; Köhrle, Josef; Grüters, Annette; Krude, Heiko; Mittag, Jens; Schöneberg, Torsten; Kleinau, Gunnar; Biebermann, Heike

    2015-06-01

    Most in vivo effects of 3-iodothyronamine (3-T1AM) have been thus far thought to be mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the 3-T1AM-induced hypothermic effect still persists in Taar1 knockout mice, which suggests additional receptor targets. In support of this general assumption, it has previously been reported that 3-T1AM also binds to the α-2A-adrenergic receptor (ADRA2A), which modulates insulin secretion. However, the mechanism of this effect remains unclear. We tested two different scenarios that may explain the effect: the sole action of 3-T1AM at ADRA2A and a combined action of 3-T1AM at ADRA2A and TAAR1, which is also expressed in pancreatic islets. We first investigated a potential general signaling modification using the label-free EPIC technology and then specified changes in signaling by cAMP inhibition and MAPKs (ERK1/2) determination. We found that 3-T1AM induced Gi/o activation at ADRA2A and reduced the norepinephrine (NorEpi)-induced MAPK activation. Interestingly, in ADRA2A/TAAR1 hetero-oligomers, application of NorEpi resulted in uncoupling of the Gi/o signaling pathway, but it did not affect MAPK activation. However, 3-T1AM application in mice over a period of 6 days at a daily dose of 5 mg/kg had no significant effects on glucose homeostasis. In summary, we report an agonistic effect of 3-T1AM on the ADRA2A-mediated Gi/o pathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate Gi/o signaling is reduced by co-stimulation with 3-T1AM. The present study therefore points to a complex spectrum of signaling modification mediated by 3-T1AM at different G protein-coupled receptors.

  15. Infiltrating macrophages promote prostate tumorigenesis via modulating androgen receptor-mediated CCL4-STAT3 signaling.

    PubMed

    Fang, Lei-Ya; Izumi, Kouji; Lai, Kuo-Pao; Liang, Liang; Li, Lei; Miyamoto, Hiroshi; Lin, Wen-Jye; Chang, Chawnshang

    2013-09-15

    Infiltrating macrophages are a key component of inflammation during tumorigenesis, but the direct evidence of such linkage remains unclear. We report here that persistent coculturing of immortalized prostate epithelial cells with macrophages, without adding any carcinogens, induces prostate tumorigenesis and that induction involves the alteration of signaling of macrophage androgen receptor (AR)-inflammatory chemokine CCL4-STAT3 activation as well as epithelial-to-mesenchymal transition and downregulation of p53/PTEN tumor suppressors. In vivo studies further showed that PTEN(+/-) mice lacking macrophage AR developed far fewer prostatic intraepithelial neoplasia (PIN) lesions, supporting an in vivo role for macrophage AR during prostate tumorigenesis. CCL4-neutralizing antibody effectively blocked macrophage-induced prostate tumorigenic signaling and targeting AR via an AR-degradation enhancer, ASC-J9, reduced CCL4 expression, and xenografted tumor growth in vivo. Importantly, CCL4 upregulation was associated with increased Snail expression and downregulation of p53/PTEN in high-grade PIN and prostate cancer. Together, our results identify the AR-CCL4-STAT3 axis as key regulators during prostate tumor initiation and highlight the important roles of infiltrating macrophages and inflammatory cytokines for the prostate tumorigenesis.

  16. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  17. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  18. Acid evoked thermal hyperalgesia involves peripheral P2Y1 receptor mediated TRPV1 phosphorylation in a rodent model of thrombus induced ischemic pain

    PubMed Central

    2014-01-01

    Background We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH. Results Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals. Conclusion Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of

  19. Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway

    PubMed Central

    Liu, Hong-Shuai; Shi, Hai-Lian; Huang, Fei; Peterson, Karin E.; Wu, Hui; Lan, Yun-Yi; Zhang, Bei-Bei; He, Yi-Xin; Woods, Tyson; Du, Min; Wu, Xiao-Jun; Wang, Zheng-Tao

    2016-01-01

    Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases. PMID:26750705

  20. System theoretical investigation of human epidermal growth factor receptor-mediated signalling

    SciTech Connect

    Zhang, Yi; Shankaran, Harish; Opresko, Lee; Resat, Haluk

    2008-09-01

    The partitioning of biological networks into coupled functional modules is gaining increasing attention in the biological sciences. This approach has the advantage that predicting a system level response does not require a mechanistic description of the internal dynamics of each module. Identification of the input-output characteristics of the network modules and the connectivity between the modules provide the necessary quantitative representation of system dynamics. However, determination of the input-output relationships of the modules is not trivial; it requires the controlled perturbation of module inputs and systematic analysis of experimental data. In this report, we apply a system theoretical analysis approach to derive the causal input-output relationships of the functional module for the human epidermal growth factor receptor (HER) mediated Erk and Akt signaling pathways. Using a library of cell lines expressing varying levels of EGFR and HER2, we show that a transfer function-based representation can be successfully applied to quantitatively characterize information transfer in this system.

  1. The Adapter Molecule Sin Regulates T-Cell-Receptor-Mediated Signal Transduction by Modulating Signaling Substrate Availability

    PubMed Central

    Xing, Luzhou; Donlin, Laura T.; Miller, Rebecca H.; Alexandropoulos, Konstantina

    2004-01-01

    Engagement of the T-cell receptor (TCR) results in the activation of a multitude of signaling events that regulate the function of T lymphocytes. These signaling events are in turn modulated by adapter molecules, which control the final functional output through the formation of multiprotein complexes. In this report, we identified the adapter molecule Sin as a new regulator of T-cell activation. We found that the expression of Sin in transgenic T lymphocytes and Jurkat T cells inhibited interleukin-2 expression and T-cell proliferation. This inhibitory effect was specific and was due to defective phospholipase C-γ (PLC-γ) phosphorylation and activation. In contrast to other adapters that become phosphorylated upon TCR stimulation, Sin was constitutively phosphorylated in resting cells by the Src kinase Fyn and bound to signaling intermediates, including PLC-γ. In stimulated cells, Sin was transiently dephosphorylated, which coincided with transient dissociation of Fyn and PLC-γ. Downregulation of Sin expression using Sin-specific short interfering RNA oligonucleotides inhibited transcriptional activation in response to TCR stimulation. Our results suggest that endogenous Sin influences T-lymphocyte signaling by sequestering signaling substrates and regulating their availability and/or activity in resting cells, while Sin is required for targeting these intermediates to the TCR for fast signal transmission during stimulation. PMID:15121874

  2. Repeated exposure of adult rats to Aroclor 1254 induces neuronal injury and impairs the neurochemical manifestations of the NMDA receptor-mediated intracellular signaling in the hippocampus.

    PubMed

    Hilgier, Wojciech; Łazarewicz, Jerzy W; Strużynska, Lidia; Frontczak-Baniewicz, Małgorzata; Albrecht, Jan

    2012-01-01

    Aroclor 1254 is a mixture of polychlorinated biphenyls (PCBs), a class of environmental toxins which cause a wide spectrum of neurotoxic effects. Learning and memory deficits are the profound effects of PCBs which may be related to hippocampal dysfunction. To get insight into the underlying neurochemical mechanisms, we employed the microdialysis technique to investigate the effect of repeated exposure of adult male Wistar rats to Aroclor 1254 (10mg/kg b.w., daily, ig., for 14days), on the neurochemical parameters of NMDA receptor-mediated glutamatergic signaling in the hippocampus in vivo assessed using the microdialysis technique. The results demonstrated that exposure to Aroclor 1254, which was associated with substantial neuronal damage and loss in the hippocampus, markedly decreased the NMDA-induced extracellular accumulation of newly loaded (45)CaCl(2), cGMP and glutamate, and reduced the basal content of the NO precursor, arginine, indicating inhibition of the NMDA/NO/cGMP pathway. Aroclor 1254 exposure also decreased the basal microdialysate content of glutamate and glutamine, which may cause inadequate supply of the neurotransmitter glutamate, while the level of two other neuroactive amino acids, aspartate or taurine was not affected by the exposure. The results underscore neuronal lesion and inhibition of NMDA receptor-mediated glutamatergic signaling in hippocampus as a potential major contributor to the cognitive deficits associated with exposure to PCB.

  3. Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia.

    PubMed

    Szűcs, Edina; Büki, Alexandra; Kékesi, Gabriella; Horváth, Gyöngyi; Benyhe, Sándor

    2016-04-21

    Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals.

  4. Ontogeny of catecholamine and adenosine receptor-mediated cAMP signaling of embryonic red blood cells: role of cGMP-inhibited phosphodiesterase 3 and hemoglobin.

    PubMed

    Baumann, R; Blass, C; Götz, R; Dragon, S

    1999-12-15

    We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, beta-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and beta-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

  5. Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

    PubMed Central

    Song, J; Kang, S M; Kim, E; Kim, C-H; Song, H-T; Lee, J E

    2015-01-01

    In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis. PMID:26247729

  6. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling.

    PubMed

    Glinka, Andrei; Dolde, Christine; Kirsch, Nadine; Huang, Ya-Lin; Kazanskaya, Olga; Ingelfinger, Dierk; Boutros, Michael; Cruciat, Cristina-Maria; Niehrs, Christof

    2011-10-01

    R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.

  7. Endothelin-1/endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis.

    PubMed

    Teoh, Jian-peng; Park, Kyoung-mi; Wang, Yongchao; Hu, Qiuping; Kim, Sangmi; Wu, Guangyu; Huang, Shuang; Maihle, Nita; Kim, Il-man

    2014-12-01

    The endothelin-1 (ET-1)/endothelin A receptor (ETAR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ETAR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ETAR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ETAR-targeted OC therapy, we investigated the role of other G protein subunits such as Gαs in the ETAR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ETAR axis is well-characterized, Gαs signaling inhibits ETAR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ETAR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ETAR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ETAR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ETAR axis in OC.

  8. Toll-like receptor-mediated signaling cascade as a regulator of the inflammation network during alcoholic liver disease

    PubMed Central

    Ceccarelli, Sara; Nobili, Valerio; Alisi, Anna

    2014-01-01

    Chronic abuse of alcohol leads to various histological abnormalities in the liver. These are conditions collectively known as alcoholic liver disease (ALD). Currently, ALD is considered to be one of the major causes of death worldwide. An impaired intestinal barrier with related endotoxemia is among the various pathogenetic factors. This is mainly characterized by circulating levels of lipopolysaccharide (LPS), considered critical for the onset of intra-hepatic inflammation. This in turn promotes hepatocellular damage and fibrosis in ALD. Elevated levels of LPS exert their effects by binding to Toll-like receptors (TLRs) which are expressed by all liver-resident cells. The activation of TLR signaling triggers an overproduction and release of some cytokines, which promote an autocatalytic cascade of other pro-inflammatory signals. In this review, we provide an overview of the mechanisms that sustain LPS-mediated activation of TLR signaling, reporting current experimental and clinical evidence of its role during inflammation in ALD. PMID:25469012

  9. ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells.

    PubMed

    Lin, Kana; Fang, Sanhua; Cai, Beilei; Huang, Xueqin; Zhang, Xiayan; Lu, Yunbi; Zhang, Weiping; Wei, Erqing

    2014-07-01

    The CysLT2 receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT2) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT2 receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2 and pcDNA3.1-rGPR17. Leukotriene C4 (LTC4) and LTD4 were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC4 and LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT2, but not in those expressing hCysLT1 and rGPR17. In hCysLT2-HEK293 cells, LTC4 induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT2 receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC4-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT2 receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC4 and LTD4.

  10. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

    PubMed

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  11. Activation of Retinoid Receptor-Mediated Signaling Ameliorates Diabetes-Induced Cardiac Dysfunction in Zucker Diabetic Rats

    PubMed Central

    Guleria, Rakeshwar S.; Singh, Amar B.; Nizamutdinova, Irina T.; Souslova, Tatiana; Mohammad, Amin A.; Kendall, Jonathan A.; Baker, Kenneth M.; Pan, Jing

    2013-01-01

    Diabetic cardiomyopathy (DCM) is a significant contributor to the morbidity and mortality associated with diabetes and metabolic syndrome. Retinoids, through activation of retinoic acid receptor (RAR) and retinoid×receptor (RXR), have been linked to control of glucose and lipid homeostasis, with effects on obesity and diabetes. However, the functional role of RAR and RXR in the development of DCM remains unclear. Zucker diabetic fatty (ZDF) and lean rats were treated with Am580 (RARα agonist) or LGD1069 (RXR agonist) for 16 weeks, and cardiac function and metabolic alterations were determined. Hyperglycemia, hyperlipidemia and insulin resistance were observed in ZDF rats. Diabetic cardiomyopathy was characterized in ZDF rats by increased oxidative stress, apoptosis, fibrosis, inflammation, activation of MAP kinases and NF-κB signaling and diminished Akt phosphorylation, along with decreased glucose transport and increased cardiac lipid accumulation, and ultimately diastolic dysfunction. Am580 and LGD1069 attenuated diabetes-induced cardiac dysfunction and the pathological alterations, by improving glucose tolerance and insulin resistance; facilitating Akt activation and glucose utilization, and attenuating oxidative stress and interrelated MAP kinase and NF-κB signaling pathways. Am580 inhibited body weight gain, attenuated the increased cardiac fatty acid uptake, β-oxidation and lipid accumulation in the hearts of ZDF rats. However, LGD1069 promoted body weight gain, hyperlipidemia and cardiac lipid accumulation. In conclusion, our data suggest that activation of RAR and RXR may have therapeutic potential in the treatment of diabetic cardiomyopathy. However, further studies are necessary to clarify the role of RAR and RXR in the regulation of lipid metabolism and homeostasis. PMID:23395853

  12. Activation of retinoid receptor-mediated signaling ameliorates diabetes-induced cardiac dysfunction in Zucker diabetic rats.

    PubMed

    Guleria, Rakeshwar S; Singh, Amar B; Nizamutdinova, Irina T; Souslova, Tatiana; Mohammad, Amin A; Kendall, Jonathan A; Baker, Kenneth M; Pan, Jing

    2013-04-01

    Diabetic cardiomyopathy (DCM) is a significant contributor to the morbidity and mortality associated with diabetes and metabolic syndrome. Retinoids, through activation of retinoic acid receptor (RAR) and retinoid x receptor (RXR), have been linked to control glucose and lipid homeostasis, with effects on obesity and diabetes. However, the functional role of RAR and RXR in the development of DCM remains unclear. Zucker diabetic fatty (ZDF) and lean rats were treated with Am580 (RARα agonist) or LGD1069 (RXR agonist) for 16 weeks, and cardiac function and metabolic alterations were determined. Hyperglycemia, hyperlipidemia and insulin resistance were observed in ZDF rats. Diabetic cardiomyopathy was characterized in ZDF rats by increased oxidative stress, apoptosis, fibrosis, inflammation, activation of MAP kinases and NF-κB signaling and diminished Akt phosphorylation, along with decreased glucose transport and increased cardiac lipid accumulation, and ultimately diastolic dysfunction. Am580 and LGD1069 attenuated diabetes-induced cardiac dysfunction and the pathological alterations, by improving glucose tolerance and insulin resistance; facilitating Akt activation and glucose utilization, and attenuating oxidative stress and interrelated MAP kinase and NF-κB signaling pathways. Am580 inhibited body weight gain, attenuated the increased cardiac fatty acid uptake, β-oxidation and lipid accumulation in the hearts of ZDF rats. However, LGD1069 promoted body weight gain, hyperlipidemia and cardiac lipid accumulation. In conclusion, our data suggest that activation of RAR and RXR may have therapeutic potential in the treatment of diabetic cardiomyopathy. However, further studies are necessary to clarify the role of RAR and RXR in the regulation of lipid metabolism and homeostasis.

  13. Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa.

    PubMed

    Nixon, Brett; Paul, Jonathan W; Spiller, Cassy M; Attwell-Heap, Abigail G; Ashman, Leonie K; Aitken, R John

    2005-10-15

    Mammalian spermatozoa must become ;capacitated' in the female reproductive tract before they gain the ability to fertilize the oocyte. The attainment of a capacitated state has been correlated with a number of biochemical changes, the most notable of which is a dramatic increase in the tyrosine phosphorylation status of these cells. Despite its biological importance, the mechanisms responsible for initiating this tyrosine phosphorylation cascade in vivo are unknown. Here, we report that this signalling pathway can be elicited in a rapid, dose-dependent and lectin-specific manner by wheat germ agglutinin (WGA), but none of 18 other lectins assessed. This response was abrogated by prior enzymatic cleavage of either sialic acid or GlcNAc residues from the sperm surface and by treatment with a range of pharmacological inhibitors directed against protein kinase A, protein tyrosine kinases and intermediates including Src. Proteomic analysis of the WGA-binding sites on the sperm surface identified the putative cognate receptor as platelet cell adhesion molecule 1 (PECAM-1/CD31). This conclusion was supported by the following evidence: (i) anti-PECAM-1 antibodies identified a molecule of the correct molecular mass in human spermatozoa, (ii) PECAM-1 could be isolated from a pool of sperm surface proteins using WGA immobilized on a solid phase support, (iii) PECAM-1 and WGA co-localized to the sperm surface and (iv) anti-PECAM-1 antibodies could completely block the ability of WGA to stimulate tyrosine phosphorylation in these cells. Collectively, these data provide the first evidence that a receptor-mediated signal transduction pathway triggers human sperm capacitation and identifies PECAM-1 as the probable initiator of this second messenger cascade. PMID:16219692

  14. CB1 Receptor-Mediated Signaling Underlies the Hippocampal Synaptic, Learning and Memory Deficits Following Treatment with JWH-081, a New Component of Spice/K2 Preparations

    PubMed Central

    Basavarajappa, Balapal S.; Subbanna, Shivakumar

    2014-01-01

    Recently, synthetic cannabinoids have been sprayed onto plant material, which is subsequently packaged and sold as “Spice” or “K2” to mimic the effects of marijuana. A recent report identified several synthetic additives in samples of “Spice/K2”, including JWH-081, a synthetic ligand for the cannabinoid receptor 1 (CB1). The deleterious effects of JWH-081 on brain function are not known, particularly on CB1 signaling, synaptic plasticity, learning and memory. Here, we evaluated the effects of JWH-081 on pCaMKIV, pCREB and pERK1/2 signaling events followed by long-term potentiation (LTP), hippocampal-dependent learning and memory tasks using CB1 receptor wild type (WT) and knockout (KO) mice. Acute administration of JWH-081 impaired CaMKIV phosphorylation in a dose-dependent manner, whereas inhibition of CREB phosphorylation in CB1 receptor WT mice was observed only at higher dose of JWH-081 (1.25 mg/kg). JWH-081 at higher dose impaired CaMKIV and CREB phosphorylation in a time –dependent manner in CB1 receptor WT mice but not in KO mice and failed to alter ERK1/2 phosphorylation. In addition, SR treated or CB1 receptor KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio compared to vehicle or WT littermates. In hippocampal slices, JWH-081 impaired LTP in CB1 receptor WT but not in KO littermates. Furthermore, JWH-081 at higher dose impaired object recognition, spontaneous alternation and spatial memory on the Y-maze in CB1 receptor WT mice but not in KO mice. Collectively our findings suggest that deleterious effects of JWH-081 on hippocampal function involves CB1 receptor mediated impairments in CaMKIV and CREB phosphorylation, LTP, learning and memory in mice. PMID:24123667

  15. Insulin receptor-mediated nutritional signalling regulates juvenile hormone biosynthesis and vitellogenin production in the German cockroach.

    PubMed

    Abrisqueta, Marc; Süren-Castillo, Songül; Maestro, José L

    2014-06-01

    Female reproductive processes, which comprise, amongst others, the synthesis of yolk proteins and the endocrine mechanisms which regulate this synthesis, need a considerable amount of energy and resources. The role of communicating that the required nutritional status has been attained is carried out by nutritional signalling pathways and, in particular, by the insulin receptor (InR) pathway. In the present study, using the German cockroach, Blattella germanica, as a model, we analysed the role of InR in different processes, but mainly those related to juvenile hormone (JH) synthesis and vitellogenin production. We first cloned the InR cDNA from B. germanica (BgInR) and then determined that its expression levels were constant in corpora allata and fat body during the first female gonadotrophic cycle. Results showed that the observed increase in BgInR mRNA in fat body from starved compared to fed females was abolished in those females treated with systemic RNAi in vivo against the transcription factor BgFoxO. RNAi-mediated BgInR knockdown during the final two nymphal stages produced significant delays in the moults, together with smaller adult females which could not spread the fore- and hindwings properly. In addition, BgInR knockdown led to a severe inhibition of juvenile hormone synthesis in adult female corpora allata, with a concomitant reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, HMG-CoA reductase and methyl farnesoate epoxidase. BgInR RNAi treatment also reduced fat body vitellogenin mRNA and oocyte growth. Our results show that BgInR knockdown produces similar phenotypes to those obtained in starved females in terms of corpora allata activity and vitellogenin synthesis, and indicate that the InR pathway mediates the activation of JH biosynthesis and vitellogenin production elicited by nutrition signalling. PMID:24657890

  16. Signal transduction pathways involved in kinin B2 receptor-mediated vasodilation in the rat isolated perfused kidney

    PubMed Central

    Bagaté, Karim; Grima, Michèle; Imbs, Jean-Louis; Jong, Wybren De; Helwig, Jean-Jacques; Barthelmebs, Mariette

    2001-01-01

    The signal transduction pathways involved in kinin B2 receptor-related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F2α or KCl-induced constriction, the vasodilator response to a selective B2 receptor agonist, Tyr(Me)8bradykinin (Tyr(Me)8BK), was assessed.Tyr(Me)8BK produced a concentration- and endothelium-dependent relaxation that was decreased by about 30 – 40% after inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine (L-NOARG) or of cyclo-oxygenase by indomethacin; a greater decrease (about 40 – 50%) was observed after concomitant inhibition of the two pathways.High extracellular K+ diminished Tyr(Me)8BK-induced relaxation by about 75% suggesting a major contribution of endothelium-derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo-oxygenase inhibition. The K+ channel inhibitors, tetrabutylammonium (non-specific) and charybdotoxin (specific for Ca2+-activated K+ channel), suppressed Tyr(Me)8BK-induced relaxation resistant to L-NOARG and indomethacin.Inhibition of cytochrome P450 (clotrimazole or 7-ethoxyresorufin) decreased the NO/prostanoids-independent relaxation to Tyr(Me)8BK by more than 60%, while inhibition of the cannabinoid CB1 receptor (SR 141716A) had only a moderate effect.Acetylcholine induced a concentration-dependent relaxation with characteristics nearly similar to the response to Tyr(Me)8BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L-NOARG or removal of endothelium) but remained unchanged otherwise.These results indicate that the activation of kinin B2 receptors in the rat isolated kidney elicits an endothelium-dependent vasorelaxation, mainly dependent on the activation of charybdotoxin-sensitive Ca2+-activated K+ channels. In addition, cytochrome P450 derivatives appear to be involved. PMID:11309245

  17. Cyproheptadine enhances the I(K) of mouse cortical neurons through sigma-1 receptor-mediated intracellular signal pathway.

    PubMed

    He, Yan-Lin; Zhang, Chun-Lei; Gao, Xiao-Fei; Yao, Jin-Jing; Hu, Chang-Long; Mei, Yan-Ai

    2012-01-01

    Cyproheptadine (CPH) is a histamine- and serotonin-receptor antagonist, and its effects are observed recently in the modulation of multiple intracellular signals. In this study, we used cortical neurons and HEK-293 cells transfected with Kv2.1 α-subunit to address whether CPH modify neural voltage-gated K(+) channels by a mechanism independent of its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the I(K) by reducing the activity of protein kinas A (PKA). Inhibition of G(i) eliminated the CPH-induced effect on both the I(K) and PKA. Blocking of 5-HT-, M-, D(2)-, H(1)- or H(2)-type GPCR receptors with relevant antagonists did not eliminate the CPH-induced effect on the I(K). Antagonists of the sigma-1 receptor, however, blocked the effect of CPH. Moreover, the inhibition of sigma-1 by siRNA knockdown significantly reduced the CPH-induced effect on the I(K). On the contrary, sigma-1 receptor agonist mimicked the effects of CPH on the induction of I(K). A ligand-receptor binding assay indicated that CPH bound to the sigma-1 receptor. Similar effect of CPH were obtained from HEK-293 cells transfected with the α-subunit of Kv2.1. In overall, we reveal for the first time that CPH enhances the I(K) by modulating activity of PKA, and that the associated activation of the sigma-1 receptor/G(i)-protein pathway might be involved. Our findings illustrate an uncharacterized effect of CPH on neuron excitability through the I(K), which is independent of histamine H(1) and serotonin receptors.

  18. Modulatory effect of insulin on T cell receptor mediated calcium signaling is blunted in long lasting type 1 diabetes mellitus.

    PubMed

    Demkow, Urszula; Winklewski, Paweł; Ciepiela, Olga; Popko, Katarzyna; Lipińska, Anna; Kucharska, Anna; Michalska, Beata; Wąsik, Maria

    2012-01-01

    Insulin significantly influences Ca(2+) signals evoked by various stimulants. In type 1 recent onset diabetes mellitus the proliferative response of T cells is significantly decreased. The number of clinical trials exploring the role of anti-CD3 monoclonal antibodies (mAb) as a therapeutic agent in recent onset diabetes mellitus type 1 is increasing last years. Therefore, a better understanding of the interplay between T cell receptor (TCR) dependent Ca(2+) increase, and insulin is of vital clinical significance. The aim of the study was to assess the effect of insulin on TCR evoked Ca(2+) responses in T lymphocytes obtained from healthy volunteers and patients suffering from long lasting diabetes mellitus type 1. Analysis was performed with use of the flow cytometer. We demonstrated that T cells ability to mobilize Ca(2+) was significantly reduced in long lasting diabetes mellitus type 1. Ca(2+) decrease achieved by the long term incubation with anti-CD3 mAb in T cells from healthy volunteers was restored by insulin. Strong interrelationship between baseline Ca(2+) level and plateau phase response to TCR stimulation was observed in the cytoplasm of cells pre-incubated with insulin from both healthy subjects and diabetic patients (r = 0.95, p < 0.0001 and r = 0.94, p < 0.0001, respectively). We postulate the existence of the interplay between TCR mediated activation and insulin. The TCR-insulin interplay is blunted in long lasting diabetes mellitus type 1. These observations may have an important implication for future therapeutic options in diabetes.

  19. Characterization of Receptor-Mediated Signal Transduction by Escherichia coli Type IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84

    PubMed Central

    Wimer-Mackin, Susan; Holmes, Randall K.; Wolf, Anne A.; Lencer, Wayne I.; Jobling, Michael G.

    2001-01-01

    Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl− secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl− secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl− secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl− secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli. PMID:11705889

  20. Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling.

    PubMed

    Kumagai, Shohei; Matsui, Kazuki; Kawaguchi, Haruyo; Yamashita, Tomomi; Mohri, Tomomi; Fujio, Yasushi; Nakayama, Hiroyuki

    2013-08-01

    Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R-MAPK signaling pathway. PMID:23867818

  1. Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons.

    PubMed

    Yoon, Dong-Hoon; Yoon, Sehyoun; Kim, Donghoon; Kim, Hyun; Baik, Ja-Hyun

    2015-01-23

    Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability. PMID:25483619

  2. P2Y₁ receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion.

    PubMed

    Wuttke, Anne; Idevall-Hagren, Olof; Tengholm, Anders

    2013-04-01

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

  3. Physical and functional association of the cbl protooncogen product with an src-family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor-mediated signaling

    PubMed Central

    1996-01-01

    To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two- hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c-cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c- cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors PMID:8627181

  4. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

    PubMed

    Avanzato, D; Genova, T; Fiorio Pla, A; Bernardini, M; Bianco, S; Bussolati, B; Mancardi, D; Giraudo, E; Maione, F; Cassoni, P; Castellano, I; Munaron, L

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1-10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  5. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

    PubMed Central

    Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; Castellano, I.; Munaron, L.

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  6. The Novel Functions of the PLC/PKC/PKD Signaling Axis in G Protein-Coupled Receptor-Mediated Chemotaxis of Neutrophils

    PubMed Central

    Xu, Xuehua; Jin, Tian

    2015-01-01

    Chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by the G protein-coupled receptor (GPCR) signaling pathway. Extracellular stimuli trigger activation of the PLC/PKC/PKD signaling axis, which controls several signaling pathways. Here, we concentrate on the novel functions of PLC/PKC/PKD signaling in GPCR-mediated chemotaxis of neutrophils. PMID:26605346

  7. RdgBα reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors.

    PubMed

    Cockcroft, Shamshad; Garner, Kathryn; Yadav, Shweta; Gomez-Espinoza, Evelyn; Raghu, Padinjat

    2016-02-01

    Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

  8. Homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system at ER-PM junctions.

    PubMed

    Chang, Chi-Lun; Liou, Jen

    2016-08-01

    The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-Ca(2+) signaling system is important for cell activation in response to various extracellular stimuli. This signaling system is initiated by receptor-induced hydrolysis of PI(4,5)P2 in the plasma membrane (PM) to generate the soluble second messenger inositol 1,4,5-trisphosphate (IP3). IP3 subsequently triggers the release of Ca(2+) from the endoplasmic reticulum (ER) store to the cytosol to activate Ca(2+)-mediated responses, such as secretion and proliferation. The consumed PM PI(4,5)P2 and ER Ca(2+) must be quickly restored to sustain signaling responses, and to maintain the homeostasis of PI(4,5)P2 and Ca(2+). Since phosphatidylinositol (PI), the precursor lipid for PM PI(4,5)P2, is synthesized in the ER membrane, and a Ca(2+) influx across the PM is required to refill the ER Ca(2+) store, efficient communications between the ER and the PM are critical for the homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system. This review describes the major findings that established the framework of the PI(4,5)P2-Ca(2+) signaling system, and recent discoveries on feedback control mechanisms at ER-PM junctions that sustain the PI(4,5)P2-Ca(2+) signaling system. Particular emphasis is placed on the characterization of ER-PM junctions where efficient communications between the ER and the PM occur, and the activation mechanisms of proteins that dynamically localize to ER-PM junctions to provide the feedback control during PI(4,5)P2-Ca(2+) signaling, including the ER Ca(2+) sensor STIM1, the extended synaptotagmin E-Syt1, and the PI transfer protein Nir2. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

  9. SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

    PubMed Central

    2012-01-01

    Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains. PMID:22974441

  10. P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat cortical astrocytes.

    PubMed

    Jacob, Pedro F; Vaz, Sandra H; Ribeiro, Joaquim A; Sebastião, Ana M

    2014-08-01

    Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca(2+) ]i . Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca(2+) levels through the PLC-PKC pathway. GABA transporters are a substrate for a number of Ca(2+) -related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial-enriched cultures expressed functional P2Y receptors, as evaluated through Ca(2+) imaging, being P2Y1 the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT-1 or GAT-3 transporters, decreasing the Vmax kinetic constant. ATP-induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine-mediated effect. This, was mimicked by a specific agonist for the P2Y1,12,13 receptor (2-MeSADP). The effect of 2-MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y1 selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2-MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA-AM) or where calcium stores were depleted (α-cyclopiazonic acid, CPA). In conclusion, P2Y1 receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y1 -mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. PMID:24733747

  11. Peripheral μ-opioid receptor mediated inhibition of calcium signaling and action potential-evoked calcium fluorescent transients in primary afferent CGRP nociceptive terminals.

    PubMed

    Baillie, Landon D; Schmidhammer, Helmut; Mulligan, Sean J

    2015-06-01

    While μ-opioid receptor (MOR) agonists remain the most powerful analgesics for the treatment of severe pain, serious adverse side effects that are secondary to their central nervous system actions pose substantial barriers to therapeutic use. Preclinical and clinical evidence suggest that peripheral MORs play an important role in opioid analgesia, particularly under inflammatory conditions. However, the mechanisms of peripheral MOR signaling in primary afferent pain fibres remain to be established. We have recently introduced a novel ex vivo optical imaging approach that, for the first time, allows the study of physiological functioning within individual peripheral nociceptive fibre free nerve endings in mice. In the present study, we found that MOR activation in selectively identified, primary afferent CGRP nociceptive terminals caused inhibition of N-type Ca(2+) channel signaling and suppression of action potential-evoked Ca(2+) fluorescent transients mediated by 'big conductance' Ca(2+)-activated K(+) channels (BKCa). In the live animal, we showed that the peripherally acting MOR agonist HS-731 produced analgesia and that BKCa channels were the major effectors of the peripheral MOR signaling. We have identified two key molecular transducers of MOR activation that mediate significant inhibition of nociceptive signaling in primary afferent terminals. Understanding the mechanisms of peripheral MOR signaling may promote the development of pathway selective μ-opioid drugs that offer improved therapeutic profiles for achieving potent analgesia while avoiding serious adverse central side effects. PMID:25721395

  12. Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry

    PubMed Central

    Park, Yurim; Lee, Seung-Hyun; Jo, Su-Hyun; Chung, Sungkwon; Kim, Kyong-Tai

    2016-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling. PMID:26963511

  13. Purinergic signaling via P2Y receptors up-mediates IL-6 production by liver macrophages/Kupffer cells.

    PubMed

    Ishimaru, Makiko; Yusuke, Negishi; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Takenouchi, Takato; Kitani, Hiroshi; Kojima, Shuji

    2014-06-01

    Resident macrophages in the liver (Kupffer cells) produce various cytokines and chemokines, and have important roles in hepatitis and liver fibrosis. The cells are activated by various factors, for example lipopolysaccharide (LPS), which is an endotoxin and is high in the blood of patients with liver cirrhosis. Involvement of P2 receptors in the release of pro-inflammatory cytokines from Kupffer cells is little. In this study, we investigated purinergic signaling in the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, from liver Kupffer cells of C57BL/6 mice (KUP5 cells). KUP5cells were isolated from C57BL/6 mice and cultivated with Dulbecco's modified Eagle's medium. The cells were stimulated with LPS. LPS-induced IL-6 production by KUP5 cells was suppressed significantly by pretreatments with non-selective P2 antagonist suramin, P2Y13antagonist MRS2211, and ecto-nucleotidase, whereas P2Y receptor agonists, significantly increased the IL-6 production. P2Y13knockdown reduced LPS-induced IL-6 production, but by less than 50%. These results would suggest that P2Y receptors including P2Y13and others, may involves in LPS-induced IL-6 production in Kupffer cells, leading to the liver inflammation. Therefore, we first showed the importance of purinergic signaling via P2Y receptors in the activation of Kupffer cells and liver injury, which is worthwhile in drug development for liver diseases. PMID:24849676

  14. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

    PubMed

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  15. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    PubMed Central

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-01-01

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation. PMID:22909302

  16. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  17. Chronic suppression of inositol 1,4,5-triphosphate receptor-mediated calcium signaling in cerebellar purkinje cells alleviates pathological phenotype in spinocerebellar ataxia 2 mice.

    PubMed

    Kasumu, Adebimpe W; Liang, Xia; Egorova, Polina; Vorontsova, Daria; Bezprozvanny, Ilya

    2012-09-12

    Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder characterized by progressive ataxia. SCA2 results from a poly(Q) (polyglutamine) expansion in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are primarily affected in SCA2, but the cause of PC dysfunction and death in SCA2 is poorly understood. In previous studies, we reported that mutant but not wild-type Atx2 specifically binds the inositol 1,4,5-trisphosphate receptor (InsP(3)R) and increases its sensitivity to activation by InsP3. We further proposed that the resulting supranormal calcium (Ca2+) release from the PC endoplasmic reticulum plays a key role in the development of SCA2 pathology. To test this hypothesis, we achieved a chronic suppression of InsP(3)R-mediated Ca2+ signaling by adenoassociated virus-mediated expression of the inositol 1,4,5-phosphatase (Inpp5a) enzyme (5PP) in PCs of a SCA2 transgenic mouse model. We determined that recombinant 5PP overexpression alleviated age-dependent dysfunction in the firing pattern of SCA2 PCs. We further discovered that chronic 5PP overexpression also rescued age-dependent motor incoordination and PC death in SCA2 mice. Our findings further support the important role of supranormal Ca2+ signaling in SCA2 pathogenesis and suggest that partial inhibition of InsP3-mediated Ca2+ signaling could provide therapeutic benefit for the patients afflicted with SCA2 and possibly other SCAs.

  18. Inositol-1,4,5-triphosphate receptors mediate activity-induced synaptic Ca2+ signals in muscle fibers and Ca2+ overload in slow-channel syndrome.

    PubMed

    Zayas, Roberto; Groshong, Jason S; Gomez, Christopher M

    2007-04-01

    Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca2+ overload in SCS we studied the role of sarcoplasmic Ca2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca2+ accumulates around wild type NMJ and discovered that Ca2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca2+ and that inositol-1,4,5-triphosphate receptors (IP3R), and to a lesser degree L-type voltage gated Ca2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca2+, and IP3R is responsible for localized Ca2+ signals observed in muscle fibers and the Ca2+ overload observed in SCS.

  19. PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells

    PubMed Central

    Holighaus, Yvonne; Mustafa, Tomris; Eiden, Lee E.

    2011-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP’s homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca2+. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders. PMID:21693142

  20. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    SciTech Connect

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok; Kang, Ho Young; Kim, Manbok; Koh, Sang Seok; Chung, Young-Hwa

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  1. Single-walled carbon nanotube exposure induces membrane rearrangement and suppression of receptor-mediated signalling pathways in model mast cells

    PubMed Central

    Umemoto, Eric Y.; Speck, Mark; Shimoda, Lori M.N.; Kahue, Kara; Sung, Carl; Stokes, Alexander J.; Turner, Helen

    2014-01-01

    Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and pro-inflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified ‘interactome’ isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism. PMID:24910985

  2. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling.

    PubMed

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok; Kang, Ho Young; Kim, Manbok; Koh, Sang Seok; Chung, Young-Hwa

    2015-04-01

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.

  3. Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release.

    PubMed

    Cook, Zara C; Gray, Michael A; Cann, Martin J

    2012-07-27

    Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.

  4. Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

    PubMed Central

    Cai, Xiang; Flores-Hernandez, Jorge; Feng, Jian; Yan, Zhen

    2002-01-01

    Emerging evidence has implicated a potential role for 5-HT4 receptors in cognition and anxiolysis. One of the main target structures of 5-HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5-HT4 receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network. PMID:11986365

  5. Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.

    PubMed

    Akimova, Olga A; Grygorczyk, Alexandra; Bundey, Richard A; Bourcier, Nathalie; Gekle, Michael; Insel, Paul A; Orlov, Sergei N

    2006-10-20

    In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP > 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP > UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively

  6. Molecular mechanisms of calcium-sensing receptor-mediated calcium signaling in the modulation of epithelial ion transport and bicarbonate secretion.

    PubMed

    Xie, Rui; Dong, Xiao; Wong, Chase; Vallon, Volker; Tang, Bo; Sun, Jun; Yang, Shiming; Dong, Hui

    2014-12-12

    Epithelial ion transport is mainly under the control of intracellular cAMP and Ca(2+) signaling. Although the molecular mechanisms of cAMP-induced epithelial ion secretion are well defined, those induced by Ca(2+) signaling remain poorly understood. Because calcium-sensing receptor (CaSR) activation results in an increase in cytosolic Ca(2+) ([Ca(2+)]cyt) but a decrease in cAMP levels, it is a suitable receptor for elucidating the mechanisms of [Ca(2+)]cyt-mediated epithelial ion transport and duodenal bicarbonate secretion (DBS). CaSR proteins have been detected in mouse duodenal mucosae and human intestinal epithelial cells. Spermine and Gd(3+), two CaSR activators, markedly stimulated DBS without altering duodenal short circuit currents in wild-type mice but did not affect DBS and duodenal short circuit currents in cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice. Clotrimazole, a selective blocker of intermediate conductance Ca(2+)-activated K(+) channels but not chromanol 293B, a selective blocker of cAMP-activated K(+) channels (KCNQ1), significantly inhibited CaSR activator-induced DBS, which was similar in wild-type and KCNQ1 knockout mice. HCO3 (-) fluxes across epithelial cells were activated by a CFTR activator, but blocked by a CFTR inhibitor. CaSR activators induced HCO3 (-) fluxes, which were inhibited by a receptor-operated channel (ROC) blocker. Moreover, CaSR activators dose-dependently raised cellular [Ca(2+)]cyt, which was abolished in Ca(2+)-free solutions and inhibited markedly by selective CaSR antagonist calhex 231, and ROC blocker in both animal and human intestinal epithelial cells. Taken together, CaSR activation triggers Ca(2+)-dependent DBS, likely through the ROC, intermediate conductance Ca(2+)-activated K(+) channels, and CFTR channels. This study not only reveals that [Ca(2+)]cyt signaling is critical to modulate DBS but also provides novel insights into the molecular mechanisms of CaSR-mediated Ca(2+)-induced

  7. Time-dependent effects of repeated THC treatment on dopamine D2/3 receptor-mediated signalling in midbrain and striatum.

    PubMed

    Tournier, Benjamin B; Tsartsalis, Stergios; Dimiziani, Andrea; Millet, Philippe; Ginovart, Nathalie

    2016-09-15

    This study examined the time-course of alterations in levels and functional sensitivities of dopamine D2/3 receptors (D2/3R) during the course and up to 6 weeks following cessation of chronic treatment with Delta(9)-Tetrahydrocannabinol (THC) in rats. THC treatment led to an increase in D2/3R levels in striatum, as assessed using [(3)H]-(+)-PHNO, that was readily observable after one week of treatment, remained stably elevated during the subsequent 2 weeks of treatment, but fully reversed within 2 weeks of THC discontinuation. THC-induced D2/3R alterations were more pronounced and longer lasting in the dopamine cell body regions of the midbrain, wherein [(3)H]-(+)-PHNO binding was still elevated at 2 weeks but back to control values at 6 weeks after THC cessation. Parallel analyses of the psychomotor effects of pre- and post-synaptic doses of quinpirole also showed a pattern of D2/3R functional supersensitivity indicative of more rapid subsidence in striatum than in midbrain following drug cessation. These results indicate that chronic THC is associated with a biochemical and functional sensitization of D2/3R signaling, that these responses show a region-specific temporal pattern and are fully reversible following drug discontinuation. These results suggest that an increased post-synaptic D2/3R function and a decreased DA presynaptic signaling, mediated by increased D2/3R autoinhibition, may predominate during distinct phases of withdrawal and may contribute both to the mechanisms leading to relapse and to cannabinoid withdrawal symptoms. The different rates of normalization of D2/3R function in striatum and midbrain may be critical information for the development of new pharmacotherapies for cannabis dependence. PMID:27233824

  8. A Carma1/MALT1-dependent, Bcl10-independent, pathway regulates antigen receptor-mediated mTOR signaling in T cells

    PubMed Central

    Hamilton, Kristia S.; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P.

    2015-01-01

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4+ T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR. PMID:24917592

  9. Time-dependent effects of repeated THC treatment on dopamine D2/3 receptor-mediated signalling in midbrain and striatum.

    PubMed

    Tournier, Benjamin B; Tsartsalis, Stergios; Dimiziani, Andrea; Millet, Philippe; Ginovart, Nathalie

    2016-09-15

    This study examined the time-course of alterations in levels and functional sensitivities of dopamine D2/3 receptors (D2/3R) during the course and up to 6 weeks following cessation of chronic treatment with Delta(9)-Tetrahydrocannabinol (THC) in rats. THC treatment led to an increase in D2/3R levels in striatum, as assessed using [(3)H]-(+)-PHNO, that was readily observable after one week of treatment, remained stably elevated during the subsequent 2 weeks of treatment, but fully reversed within 2 weeks of THC discontinuation. THC-induced D2/3R alterations were more pronounced and longer lasting in the dopamine cell body regions of the midbrain, wherein [(3)H]-(+)-PHNO binding was still elevated at 2 weeks but back to control values at 6 weeks after THC cessation. Parallel analyses of the psychomotor effects of pre- and post-synaptic doses of quinpirole also showed a pattern of D2/3R functional supersensitivity indicative of more rapid subsidence in striatum than in midbrain following drug cessation. These results indicate that chronic THC is associated with a biochemical and functional sensitization of D2/3R signaling, that these responses show a region-specific temporal pattern and are fully reversible following drug discontinuation. These results suggest that an increased post-synaptic D2/3R function and a decreased DA presynaptic signaling, mediated by increased D2/3R autoinhibition, may predominate during distinct phases of withdrawal and may contribute both to the mechanisms leading to relapse and to cannabinoid withdrawal symptoms. The different rates of normalization of D2/3R function in striatum and midbrain may be critical information for the development of new pharmacotherapies for cannabis dependence.

  10. G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1,4,5-triphosphate receptor-mediated Ca2+ signal regulation.

    PubMed

    Zhang, Songbai; Hisatsune, Chihiro; Matsu-Ura, Toru; Mikoshiba, Katsuhiko

    2009-10-16

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated calcium (Ca(2+)) release channel and plays important roles in regulation of numerous Ca(2+)-dependent cellular responses. Many intracellular modulators and IP(3)R-binding proteins regulate the IP(3)R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP(3)R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP(3)R. The interaction was favored by the cytosolic Ca(2+) concentration and it functionally inhibited IP(3)R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP(3)R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP(3)R-mediated Ca(2+) signal through a direct interaction with IP(3)R in a cytosolic Ca(2+)-dependent manner.

  11. P2X receptors.

    PubMed

    North, R Alan

    2016-08-01

    Extracellular adenosine 5'-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2X receptors have a widespread tissue distribution. On some smooth muscle cells, P2X receptors mediate the fast excitatory junction potential that leads to depolarization and contraction. In the central nervous system, activation of P2X receptors allows calcium to enter neurons and this can evoke slower neuromodulatory responses such as the trafficking of receptors for the neurotransmitter glutamate. In primary afferent nerves, P2X receptors are critical for the initiation of action potentials when they respond to ATP released from sensory cells such as taste buds, chemoreceptors or urothelium. In immune cells, activation of P2X receptors triggers the release of pro-inflammatory cytokines such as interleukin 1β. The development of selective blockers of different P2X receptors has led to clinical trials of their effectiveness in the management of cough, pain, inflammation and certain neurodegenerative diseases.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377721

  12. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals.

    PubMed

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-07-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  13. Differential effects of amisulpride and haloperidol on dopamine D2 receptor-mediated signaling in SH-SY5Y cells.

    PubMed

    Park, Sung Woo; Seo, Mi Kyoung; Cho, Hye Yeon; Lee, Jung Goo; Lee, Bong Ju; Seol, Wongi; Kim, Young Hoon

    2011-09-01

    Dopamine D(2) receptors (D(2)R) are the primary target of antipsychotic drugs and have been shown to regulate Akt/glycogen synthase kinase-3β (GSK-3β) signaling through scaffolding protein β-arrestin 2. Amisulpride, an atypical antipsychotic drug, and haloperidol, a typical antipsychotic drug, are both potent D(2)R antagonists, but their therapeutic effects differ. In the present study, we compared the effects of amisulpride and haloperidol on the β-arrestin 2-mediated Akt/GSK-3β pathway in SH-SY5Y cells. To determine whether these drugs affected neuronal morphology in SH-SY5Y cells, we investigated the effects of amisulpride and haloperidol on neurite outgrowth using immunostaining. We examined the effects of these drugs on Akt and GSK-3β and its well-known downstream regulators, cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 levels using Western blot analysis. Amisulpride, but not haloperidol, was found to enhance neurite outgrowth. Small interfering RNA (siRNA) for β-arrestin 2 knockdown blocked the increase in amisulpride-induced neurite outgrowth. Furthermore, amisulpride increased the levels of Akt and GSK-3β phosphorylation, while haloperidol had no effect. The elevation of Akt phosphorylation induced by amisulpride was reduced by β-arrestin 2 siRNA. Moreover, amisulpride effectively increased the levels of phospho-CREB, BDNF, and Bcl-2. However, haloperidol had no effect on the levels of these proteins. Additionally, wortmannin, a phosphatidylinositol 3-kinase (PI3 K) inhibitor, blocked the stimulatory effect of amisulpride on phosphorylated Akt. Together, these results suggest that regulation of the β-arrestin 2-dependent pathway via blockade of the D(2)R in SH-SY5Y cells is one mechanism underlying the neuroprotective effect of amisulpride, but not haloperidol.

  14. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals

    PubMed Central

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-01-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  15. NF-kappaB activation by depolarization of skeletal muscle cells depends on ryanodine and IP3 receptor-mediated calcium signals.

    PubMed

    Valdés, Juan Antonio; Hidalgo, Jorge; Galaz, José Luis; Puentes, Natalia; Silva, Mónica; Jaimovich, Enrique; Carrasco, M Angélica

    2007-05-01

    Depolarization of skeletal muscle cells by either high external K(+) or repetitive extracellular field potential pulses induces calcium release from internal stores. The two components of this release are mediated by either ryanodine receptors or inositol 1,4,5-trisphosphate (IP(3)) receptors and show differences in kinetics, amplitude, and subcellular localization. We have reported that the transcriptional regulators including ERKs, cAMP/Ca(2+)-response element binding protein, c-fos, c-jun, and egr-1 are activated by K(+)-induced depolarization and that their activation requires IP(3)-dependent calcium release. We presently describe the activation of the nuclear transcription factor NF-kappaB in response to depolarization by either high K(+) (chronic) or electrical pulses (fluctuating). Calcium transients of relative short duration activate an NF-kappaB reporter gene to an intermediate level, whereas long-lasting calcium increases obtained by prolonged electrical stimulation protocols of various frequencies induce maximal activation of NF-kappaB. This activation is independent of extracellular calcium, whereas calcium release mediated by either ryanodine or IP(3) receptors contribute in all conditions tested. NF-kappaB activation is mediated by IkappaBalpha degradation and p65 translocation to the nucleus. Partial blockade by N-acetyl-l-cysteine, a general antioxidant, suggests the participation of reactive oxygen species. Calcium-dependent signaling pathways such as those linked to calcineurin and PKC also contribute to NF-kappaB activation by depolarization, as assessed by blockade through pharmacological agents. These results suggest that NF-kappaB activation in skeletal muscle cells is linked to membrane depolarization and depends on the duration of elevated intracellular calcium. It can be regulated by sequential activation of calcium release mediated by the ryanodine and by IP(3) receptors. PMID:17215326

  16. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals.

    PubMed

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-07-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  17. Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

    PubMed

    Ledderose, Carola; Woehrle, Tobias; Ledderose, Stephan; Strasser, Katharina; Seist, Richard; Bao, Yi; Zhang, Jingping; Junger, Wolfgang G

    2016-09-01

    T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

  18. Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

    PubMed

    Ledderose, Carola; Woehrle, Tobias; Ledderose, Stephan; Strasser, Katharina; Seist, Richard; Bao, Yi; Zhang, Jingping; Junger, Wolfgang G

    2016-09-01

    T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia. PMID:27020575

  19. Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability.

    PubMed

    Murugan, Malavika; Harward, Stephen; Scharff, Constance; Mooney, Richard

    2013-12-18

    Mutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian ortholog FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. Here we show that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms.

  20. Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice

    PubMed Central

    Hasegawa, Nozomi; Cai, Wenqian; Jin, Huiling; Hidaka, Yuko; Prajapati, Rajesh; Umemura, Masanari; Yokoyama, Utako; Sato, Motohiko; Okumura, Satoshi; Ishikawa, Yoshihiro

    2015-01-01

    Background Atrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals. Objectives Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF. Methods and Results Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2±104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR Ca2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR and α1-AR may play important roles in the development of AF. Conclusions We have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments. PMID:26203906

  1. Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells

    PubMed Central

    Huang, Peng; Xu, Wei; Yoon, Su-In; Chen, Chongguang; Chong, Parkson Lee-Gau; Liu-Chen, Lee-Yuan

    2008-01-01

    Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5 M Na2CO3 buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane-domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30 min shifted ∼25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-β-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher-density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [35S]GTPγS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, Gαi co-immunoprecipitated with caveolin-1, which was shown to inhibit Gαi/o, and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor. PMID:17141202

  2. Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor.

    PubMed

    Xu, Pengfei; Xu, Yongteng; Hu, Bin; Wang, Jue; Pan, Rui; Murugan, Madhuvika; Wu, Long-Jun; Tang, Yamei

    2015-11-01

    Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP-P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP-P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP-P2X7R in brain damage in RBI, suggesting that inhibition of ATP-P2X7R axis might be a potential strategy for the treatment of patients with RBI. PMID:26122280

  3. Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor.

    PubMed

    Xu, Pengfei; Xu, Yongteng; Hu, Bin; Wang, Jue; Pan, Rui; Murugan, Madhuvika; Wu, Long-Jun; Tang, Yamei

    2015-11-01

    Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP-P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP-P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP-P2X7R in brain damage in RBI, suggesting that inhibition of ATP-P2X7R axis might be a potential strategy for the treatment of patients with RBI.

  4. Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular endothelium.

    PubMed

    Viana, F; de Smedt, H; Droogmans, G; Nilius, B

    1998-08-01

    Microfluorometric measurements in Fura-2-loaded single cultured human vascular endothelial cells were used to characterize the intracellular calcium [Ca2+]i responses triggered by extracellular application of adenosine 5'-triphosphate (ATP) and other nucleotides. Application of ATP or uridine 5'-triphosphate (UTP) gave rise to dose-dependent elevations of [Ca2+]i in all the cells tested. At saturating concentrations of agonist, the [Ca2+]i response was biphasic, with an early peak and a sustained plateau. Unlike peak responses, the sustained Ca2+ plateau was sensitive to removal of Ca2+ from the external medium. Mn2+ quenching revealed the presence of Ca2+ influx during the agonist-induced calcium plateau. The agonist-evoked calcium plateau was inhibited in a dose-dependent manner by the Cl-channel blocker NPPB, by the divalent cation Ni2+ and by the imidazole antimycotic econazole. Previously, these compounds have been shown to block store-operated Ca2+ entry. The two phases of the agonist-evoked [Ca2+]i response were blocked by the specific phospholipase C inhibitor U-73122 and by intracellular injection of low molecular weight heparin, suggesting the involvement of IP3-sensitive intracellular Ca2+ stores. The pharmacological profile of the response, using different nucleotides and analogues, ATP = UTP > ADP = UDP, and no responses to P2X1 and P2Y1 agonists, suggested the involvement of P2Y2 receptors. The expression of mRNA for the P2Y2 receptor was detected by RT-PCR analysis. These results indicate that P2Y2 receptors linked to intracellular Ca2+ mobilization are present in human vascular endothelial cells. The initial [Ca2+]i mobilization is followed by a phase of elevated [Ca2+]i influx.

  5. UDP-Sugars as Extracellular Signaling Molecules: Cellular and Physiologic Consequences of P2Y14 Receptor Activation

    PubMed Central

    Lazarowski, Eduardo R.

    2015-01-01

    UDP-sugars, which are indispensable for protein glycosylation reactions in cellular secretory pathways, also act as important extracellular signaling molecules. We discuss here the broadly expressed P2Y14 receptor, a G-protein–coupled receptor targeted by UDP sugars, and the increasingly diverse set of physiologic responses discovered recently functioning downstream of this receptor in many epithelia as well as in immune, inflammatory, and other cells. PMID:25829059

  6. Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2012-09-01

    Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca(2+) oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca(2+)-driven ATP oscillations, mediate the link between Ca(2+) and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X(4) receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X(4) activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on K(ATP) channel activity and PKC or PKG signaling. This study proposes that Ca(2+)-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X(4) receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca(2+) and ATP oscillations.

  7. Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model.

    PubMed

    Delekate, Andrea; Füchtemeier, Martina; Schumacher, Toni; Ulbrich, Cordula; Foddis, Marco; Petzold, Gabor C

    2014-11-19

    Astrocytic network alterations have been reported in Alzheimer's disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-β plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.

  8. Inositol triphosphate-mediated Ca2+ signals direct purinergic P2Y receptor regulation of neuronal ion channels.

    PubMed

    Zaika, Oleg; Tolstykh, Gleb P; Jaffe, David B; Shapiro, Mark S

    2007-08-15

    Purinergic P2Y receptors are one of four types of G(q/11)-coupled receptors in rat superior cervical ganglia (SCG) sympathetic neurons. In cultured SCG neurons, purinergic and bradykinin suppression of I(M) were similar in magnitude and somewhat less than that by muscarinic agonists. The effects of the P2Y receptor agonist UTP on neuronal excitability and discharge properties were studied. Under current clamp, UTP increased action potential (AP) firing in response to depolarizing current steps, depolarized the resting potential, decreased the threshold current required to fire an AP, and decreased spike-frequency adaptation. These effects were very similar to those resulting from bradykinin stimulation and not as profound as from muscarinic stimulation or full M-current blockade. We then examined the P2Y mechanism of action. Like bradykinin, but unlike muscarinic, purinergic stimulation induced rises in intracellular [Ca(2+)](i). Tests using expression of IP(3)"sponge" or IP(3) phosphatase constructs implicated IP(3) accumulation as necessary for purinergic suppression of I(M). Overexpression of wild-type or dominant-negative calmodulin (CaM) implicated Ca(2+)/CaM in the purinergic action. Both sets of results were similar to bradykinin, and opposite to muscarinic, suppression. We also examined modulation of Ca(2+) channels. As for bradykinin, purinergic stimulation did not suppress I(Ca), unless neuronal calcium sensor-1 (NCS-1) activity was blocked by a dominant-negative NCS-1 construct. Our results indicate that P2Y receptors modulate M-type channels in SCG cells via IP(3)-mediated [Ca(2+)](i) signals in concert with CaM and not by depletion of phosphatidylinositol-4, 5-biphosphate. We group purinergic P2Y and bradykinin B(2) receptors together as having a common mode of action.

  9. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD).

    PubMed

    Okada, Y; Maeno, E; Shimizu, T; Dezaki, K; Wang, J; Morishima, S

    2001-04-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl- channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl- channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl- conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNF receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma x rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by

  10. Free energy landscape of receptor-mediated cell adhesion

    NASA Astrophysics Data System (ADS)

    Yang, Tianyi; Zaman, Muhammad H.

    2007-01-01

    Receptor-mediated cell adhesion plays a critical role in cell migration, proliferation, signaling, and survival. A number of diseases, including cancer, show a strong correlation between integrin activation and metastasis. A better understanding of cell adhesion is highly desirable for not only therapeutic but also a number of tissue engineering applications. While a number of computational models and experimental studies have addressed the issue of cell adhesion to surfaces, no model or theory has adequately addressed cell adhesion at the molecular level. In this paper, the authors present a thermodynamic model that addresses receptor-mediated cell adhesion at the molecular level. By incorporating the entropic, conformational, solvation, and long- and short-range interactive components of receptors and the extracellular matrix molecules, they are able to predict adhesive free energy as a function of a number of key variables such as surface coverage, interaction distance, molecule size, and solvent conditions. Their method allows them to compute the free energy of adhesion in a multicomponent system where they can simultaneously study adhesion receptors and ligands of different sizes, chemical identities, and conformational properties. The authors' results not only provide a fundamental understanding of adhesion at the molecular level but also suggest possible strategies for designing novel biomaterials.

  11. Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts.

    PubMed

    Certal, Mariana; Vinhas, Adriana; Pinheiro, Ana Rita; Ferreirinha, Fátima; Barros-Barbosa, Aurora Raquel; Silva, Isabel; Costa, Maria Adelina; Correia-de-Sá, Paulo

    2015-11-01

    During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca(2+)]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca(2+)]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca(2+) in the extracellular fluid. The biphasic [Ca(2+)]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca(2+)]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

  12. Arranged marriage in lipid signalling? The limited choices of PtdIns(4,5)P2 in finding the right partner.

    PubMed

    Heilmann, M; Heilmann, I

    2013-09-01

    Inositol-containing phospholipids (phosphoinositides, PIs) control numerous cellular processes in eukaryotic cells. For plants, a key involvement of PIs has been demonstrated in the regulation of membrane trafficking, cytoskeletal dynamics and in processes mediating the adaptation to changing environmental conditions. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) mediates its cellular functions via binding to various alternative target proteins. Such downstream targets of PtdIns(4,5)P(2) are characterised by the possession of specific lipid-binding domains, and binding of the PtdIns(4,5)P(2) ligand exerts effects on their activity or localisation. The large number of potential alternative binding partners - and associated cellular processes - raises the question how alternative or even contrapuntal effects of PtdIns(4,5)P(2) are orchestrated to enable cellular function. This article aims to provide an overview of recent insights and new views on how distinct functional pools of PtdIns(4,5)P(2) are generated and maintained. The emerging picture suggests that PtdIns(4,5)P(2) species containing different fatty acids influence the lateral mobility of the lipids in the membrane, possibly enabling specific interactions of PtdIns(4,5)P(2) pools with certain downstream targets. PtdIns(4,5)P(2) pools with certain functions might also be defined by protein-protein interactions of PI4P 5-kinases, which pass PtdIns(4,5)P(2) only to certain downstream partners. Individually or in combination, PtdIns(4,5)P(2) species and specific protein-protein interactions of PI4P 5-kinases might contribute to the channelling of PtdIns(4,5)P(2) signals towards specific functional effects. The dynamic nature of PI-dependent signalling complexes with specific functions is an added challenge for future studies of plant PI signalling.

  13. F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

    PubMed Central

    Gu, Yanping; Wang, Congying; Li, GuangWen

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors. PMID:27385722

  14. Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-β production.

    PubMed

    Li, Ruimei; Tan, Binghe; Yan, Yan; Ma, Xiaobin; Zhang, Na; Zhang, Zhi; Liu, Mingyao; Qian, Min; Du, Bing

    2014-11-01

    Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.

  15. Platelet P2Y12 receptors enhance signalling towards procoagulant activity and thrombin generation. A study with healthy subjects and patients at thrombotic risk.

    PubMed

    van der Meijden, Paola E J; Feijge, Marion A H; Giesen, Peter L A; Huijberts, Maya; van Raak, Lisette P M; Heemskerk, Johan W M

    2005-06-01

    Activated platelets participate in arterial thrombosis by forming aggregates and potentiating the coagulation through exposure of procoagulant phosphatidylserine. The function of the two receptors for ADP, P2Y(1) and P2Y(12), is well-established in aggregation, but is incompletely understood in the platelet procoagulant response. We established that, in PRP from healthy subjects, ADP accelerated and potentiated tissue factor induced thrombin generation exclusively via stimulation of P2Y(12) and not via P2Y(1) receptors. The P2Y(12) receptors also mediated the potentiating effect of PAR-1 stimulation on thrombin generation. Furthermore, ADP enhanced in a P2Y(12)-dependent manner the Ca(2+) response induced by thrombin, which was either added externally or generated in-situ. This ADP effect was in part dependent of phosphoinositide 3-kinase and was paralleled by increased phosphatidylserine exposure. In PRP from (young) patients with either stroke or type-II diabetes, platelet-dependent thrombin generation was similarly enhanced byADP or SFLLRN as in healthy subjects. In PRP from stroke patients of older age, the P2Y(12)-mediated contribution to thrombin generation was variably reduced by two weeks of clopidogrel medication. Remaining P2Y(12) activity after medication correlated with remaining P2Y(12)-dependent P-selectin exposure, i.e. Ca(2+)-dependent secretion, likely due to incomplete antagonism of P2Y(12) receptors. Together, these results indicate that physiological platelet agonists amplify phosphatidylserine exposure and subsequent thrombin generation by release of ADP and P2Y(12)-receptor stimulation. This P2Y(12) response is accomplished by a novel Ca(2+) signalling pathway. It is similarly active in platelets from control subjects and patients at thrombotic risk. Finally, the thrombogram method is useful for measuring incomplete P2Y(12) inhibition with clopidogrel. PMID:15968399

  16. Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways.

    PubMed

    Hawkins, Phillip T; Stephens, Len R

    2016-02-01

    There are eight members of the phosphoinositide family of phospholipids in eukaryotes; PI, PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3. Receptor activation of Class I PI3Ks stimulates the phosphorylation of PI(4,5)P2 to form PI(3,4,5)P3. PI(3,4,5)P3 is an important messenger molecule that is part of a complex signalling network controlling cell growth and division. PI(3,4,5)P3 can be dephosphorylated by both 3- and 5-phosphatases, producing PI(4,5)P2 and PI(3,4)P2, respectively. There is now strong evidence that PI(3,4)P2 generated by this route does not merely represent another pathway for removal of PI(3,4,5)P3, but can act as a signalling molecule in its own right, regulating macropinocytosis, fast endophilin-mediated endocytosis (FEME), membrane ruffling, lamellipodia and invadopodia. PI(3,4)P2 can also be synthesized directly from PI4P by Class II PI3Ks and this is important for the maturation of clathrin-coated pits [clathrin-mediated endocytosis (CME)] and signalling in early endosomes. Thus PI(3,4)P2 is emerging as an important signalling molecule involved in the coordination of several specific membrane and cytoskeletal responses. Further, its inappropriate accumulation contributes to pathology caused by mutations in genes encoding enzymes responsible for its degradation, e.g. Inpp4B.

  17. Involvement of P2X7 receptor signaling on regulating the differentiation of Th17 cells and type II collagen-induced arthritis in mice

    PubMed Central

    Fan, Zhi-Dan; Zhang, Ya-Yuan; Guo, Yi-Hong; Huang, Na; Ma, Hui-Hui; Huang, Hui; Yu, Hai-Guo

    2016-01-01

    Interleukin (IL)-17 producing T helper (Th17) cells are major effector cells in the pathogenesis of rheumatoid arthritis (RA). The P2X7 receptor (P2X7R) has emerged as a potential site in the regulation of inflammation in RA but little is known of its functional role on the differentiation of Th17 cells. This study investigates the in vitro and in vivo effects of P2X7R on Th17 cell differentiation during type II collagen (CII) induced experimental arthritis model. In CII-treated dendritic cells (DCs) and DC/CD4+ T coculture system, pretreatment with pharmacological antagonists of P2X7R (Suramin and A-438079) caused strong inhibition of production of Th17-promoting cytokines (IL-1β, TGF-β1, IL-23p19 and IL-6). Exposure to CII induced the elevation of mRNAs encoding retinoic acid receptor-related orphan receptor α and γt, which were abolished by pretreatment with P2X7R antagonists. Furthermore, blocking P2X7R signaling abolished the CII-mediated increase in IL-17A. Blockade of P2X7R remarkably inhibited hind paw swelling and ameliorated pathological changes in ankle joint of the collagen-induced arthritis mice. Thus, we demonstrated a novel function for P2X7R signaling in regulating CII-induced differentiation of Th17 cells. P2X7R signaling facilitates the development of the sophisticated network of DC-derived cytokines that favors a Th17 phenotype. PMID:27775097

  18. Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

    PubMed

    Neher, Jonas J; Neniskyte, Urte; Hornik, Tamara; Brown, Guy C

    2014-09-01

    Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 μM peroxynitrite, or 50 μM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

  19. P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling.

    PubMed

    Norambuena, Andrés; Poblete, M Inés; Donoso, M Verónica; Espinoza, C Sofía; González, Alfonso; Huidobro-Toro, J Pablo

    2008-12-01

    The nucleotide P2Y(1) receptor (P2Y(1)R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1)R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1)R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1)R and obliterated the P2Y(1)R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1)R agonist, not only displaced within 4 min the P2Y(1)R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1)R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1)R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1)R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1)R to membrane rafts, highlighting the role of this microdomain in P2Y(1)R signaling.

  20. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

    PubMed Central

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, Mª Teresa

    2015-01-01

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome. PMID:26687764

  1. Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

    PubMed Central

    2013-01-01

    Background Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors. PMID

  2. NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

    PubMed

    Vivoli, Elisa; Cappon, Andrea; Milani, Stefano; Piombanti, Benedetta; Provenzano, Angela; Novo, Erica; Masi, Alessio; Navari, Nadia; Narducci, Roberto; Mannaioni, Guido; Moneti, Gloriano; Oliveira, Claudia P; Parola, Maurizio; Marra, Fabio

    2016-10-01

    Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1β (interleukin 1β). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1β was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation. PMID:27439970

  3. NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

    PubMed

    Vivoli, Elisa; Cappon, Andrea; Milani, Stefano; Piombanti, Benedetta; Provenzano, Angela; Novo, Erica; Masi, Alessio; Navari, Nadia; Narducci, Roberto; Mannaioni, Guido; Moneti, Gloriano; Oliveira, Claudia P; Parola, Maurizio; Marra, Fabio

    2016-10-01

    Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1β (interleukin 1β). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1β was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

  4. [Glutamate receptor-mediated retinal neuronal injury in experimental glaucoma].

    PubMed

    Wang, Zhong-Feng; Yang, Xiong-Li

    2016-08-25

    Glaucoma, the second leading cause of blindness, is a neurodegenerative disease characterized by optic nerve degeneration related to apoptotic death of retinal ganglion cells (RGCs). In the pathogenesis of RGC death following the onset of glaucoma, functional changes of glutamate receptors are commonly regarded as important risk factors. During the past several years, we have explored the mechanisms underlying RGC apoptosis and retinal Müller cell reactivation (gliosis) in a rat chronic ocular hypertension (COH) model. We demonstrated that elevated intraocular pressure in COH rats may induce changes of various signaling pathways, which are involved in RGC apoptosis by modulating glutamate NMDA and AMPA receptors. Moreover, we also demonstrated that over-activation of group I metabotropic glutamate receptors (mGluR I) by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir4.1 channels. In this review, incorporating our results, we discuss glutamate receptor- mediated RGC apoptosis and Müller cell gliosis in experimental glaucoma. PMID:27546508

  5. Functional expression of P2 receptors in the inner ear of chicken embryo.

    PubMed

    Galindo, Fabian; Monjaraz, Eduardo; Galicia, Salvador; Cebada, Jorge; Cortés, Celso; Flores, Amira

    2013-10-11

    The purpose of the present study was to investigate the modulation of spontaneous afferent activity by ATP during embryonic development in a preparation isolated chicken inner ear. This work was performed using multiunit and single-unit extracellular recordings from the posterior semicircular canal nerve and the basilar papilla nerve. α,β-meATP, a P2X receptor agonist, notably increased the discharge frequency of the vestibular afferents between E15 and E18, but not in the basilar papilla. In contrast, the P2Y receptor agonist UTP produced a slight increase in the discharge frequency of basilar papilla afferents, without apparent changes in the vestibular afferent activity. 2-MeSATP, a P2Y agonist, increased the basal discharge of the primary afferents in a dose-age dependent way, but when we applied the antagonist of P2Y receptor, Reactive Blue 2 (10(-4)M), the effect of 2-MeSATP decreased significantly. This was observed both in vestibule and basilar papilla. Using RT-PCR the presence of P2X₃, P2Y₁, P2Y₂ and P2Y₆ mRNA was documented in the vestibular system with more important presence during the early stage (E15) than the later stage (E21), however in the basilar papilla we found only the P2Y₁, P2Y₂ and P2Y₆ mRNA with the same temporal course as in the vestibule. These results confirm our pharmacological findings. Together this data suggests a role for P2X receptors-mediated purinergic signaling in vestibular synaptic organization. Temporal changes in P2Y receptors during development might be involved in the establishment of the endolymphatic ion composition needed for normal vestibular and auditory transduction and/or specific cellular differentiation.

  6. Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells.

    PubMed

    Qian, Xi; Zhao, Feng-Qi

    2013-03-01

    Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1.

  7. LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation

    PubMed Central

    Tan, Xiaojun; Sun, Yue; Thapa, Narendra; Liao, Yihan; Hedman, Andrew C; Anderson, Richard A

    2015-01-01

    Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression. PMID:25588945

  8. Important roles of P2Y receptors in the inflammation and cancer of digestive system

    PubMed Central

    Wan, Han-Xing; Hu, Jian-Hong; Xie, Rei; Yang, Shi-Ming; Dong, Hui

    2016-01-01

    Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future. PMID:26908460

  9. TLR-Independent and P2X7-Dependent Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in Geographic Atrophy

    PubMed Central

    Kerur, Nagaraj; Hirano, Yoshio; Tarallo, Valeria; Fowler, Benjamin J.; Bastos-Carvalho, Ana; Yasuma, Tetsuhiro; Yasuma, Reo; Kim, Younghee; Hinton, David R.; Kirschning, Carsten J.; Gelfand, Bradley D.; Ambati, Jayakrishna

    2013-01-01

    Purpose. Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes. Methods. Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7−/−, Nfkb1−/−, and Tlr23479−/− mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE. Results. Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1−/− and P2rx7−/− mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration. Conclusions. NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and

  10. Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

    PubMed Central

    Zhong, Weixia; Hutchinson, Tarun E.; Chebolu, Seetha; Darmani, Nissar A.

    2014-01-01

    Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates

  11. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila.

    PubMed

    Kanellopoulos, Alexandros K; Semelidou, Ourania; Kotini, Andriana G; Anezaki, Maria; Skoulakis, Efthimios M C

    2012-09-19

    Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

  12. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75NTR receptor-mediated activation of RhoA signaling pathways

    PubMed Central

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-01-01

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75NTR-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75NTR, Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75NTR-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75NTR-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75NTR-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75NTR/RhoA/ROCK pathway, or overexpression of a p75NTR mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75NTR/RhoA/ROCK signaling pathway. Also, our results

  13. PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways.

    PubMed

    Castorina, Alessandro; Scuderi, Soraya; D'Amico, Agata Grazia; Drago, Filippo; D'Agata, Velia

    2014-03-10

    PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating

  14. A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling.

    PubMed

    Wright, Forrest A; Lu, Justine P; Sliter, Danielle A; Dupré, Nicolas; Rouleau, Guy A; Wojcikiewicz, Richard J H

    2015-05-29

    RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca(2+) mobilization was significantly impaired, whereas neither Ca(2+) store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys(48)- and Lys(63)-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca(2+) mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.

  15. A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation

    PubMed Central

    Purvis, Jeremy E.; Chatterjee, Manash S.; Brass, Lawrence F.

    2008-01-01

    To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and Gq-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods. PMID:18596227

  16. P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides.

    PubMed

    Insel, P A; Ostrom, R S; Zambon, A C; Hughes, R J; Balboa, M A; Shehnaz, D; Gregorian, C; Torres, B; Firestein, B L; Xing, M; Post, S R

    2001-04-01

    1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways. PMID:11339212

  17. P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides.

    PubMed

    Insel, P A; Ostrom, R S; Zambon, A C; Hughes, R J; Balboa, M A; Shehnaz, D; Gregorian, C; Torres, B; Firestein, B L; Xing, M; Post, S R

    2001-04-01

    1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.

  18. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

    PubMed

    Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Ding, Biao; Li, Yi

    2016-09-01

    The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

  19. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development

    PubMed Central

    Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Li, Yi

    2016-01-01

    The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

  20. Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus.

    PubMed

    Tamm, Kristi; Merits, Andres; Sarand, Inga

    2008-03-01

    The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues (648)RRR(650) with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations. PMID:18272758

  1. Ion channel regulation by phosphoinositides analyzed with VSPs-PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility.

    PubMed

    Rjasanow, Alexandra; Leitner, Michael G; Thallmair, Veronika; Halaszovich, Christian R; Oliver, Dominik

    2015-01-01

    The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K(+) channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs.

  2. Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

    PubMed Central

    Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

    2015-01-01

    The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID

  3. Sustained Release of Prostaglandin E2 in Fibroblasts Expressing Ectopically Cyclooxygenase 2 Impairs P2Y-Dependent Ca2+-Mobilization

    PubMed Central

    Pimentel-Santillana, María; Través, Paqui G.; Pérez-Sen, Raquel; Delicado, Esmerilda G.; Martín-Sanz, Paloma; Miras-Portugal, María Teresa; Boscá, Lisardo

    2014-01-01

    The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2 in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2 when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2 was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2 involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2 reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2 is involved in the regulation of P2Y signaling in these cells. PMID:25214717

  4. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  5. P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

    PubMed Central

    Tackett, Bryan C.; Sun, Hongdan; Mei, Yu; Maynard, Janielle P.; Cheruvu, Sayuri; Mani, Arunmani; Hernandez-Garcia, Andres; Vigneswaran, Nadarajah; Karpen, Saul J.

    2014-01-01

    Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2−/−) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24–72 h) in response to 70% PH were impaired in P2Y2−/− mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2−/− remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2−/− mice were treated with ATP or ATPγS for 5–120 min and 12–24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH. PMID:25301185

  6. Possible involvement of P2X7 receptor activation in microglial neuroprotection against focal cerebral ischemia in rats.

    PubMed

    Yanagisawa, Daijiro; Kitamura, Yoshihisa; Takata, Kazuyuki; Hide, Izumi; Nakata, Yoshihiro; Taniguchi, Takashi

    2008-06-01

    Microglia play important roles in the pathogenic cascade following cerebral ischemia, since they express growth factors, chemokines and regulatory cytokines as well as free radicals and other toxic mediators. P2X7 receptor, a subtype of a family of P2 purinoceptors, is primarily expressed in microglia and macrophages, suggesting that it regulates immune function and inflammatory responses. However, the involvement of ATP in such microglial responses after cerebral ischemia is not yet understood. In this study, we investigated the possible involvement of ATP, especially through the P2X7 receptors, in a rat model of focal cerebral ischemia. In immunohistochemical analysis, P2X7 receptor-like immunoreactivity was predominantly detected in microglia, and then activated microglia accumulated in the ischemic region, in rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Intracerebroventricular injection with P2X7 receptor agonist 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) improved behavioral dysfunction accessed by rota-rod test and ischemic neural injury induced by MCAO. In contrast, P2X7 receptor antagonist adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP) exacerbated ischemic brain damage. These results suggest that microglia play an important role in neuroprotection against rat cerebral ischemia, which is regulated by a P2X7 receptor-mediated ATP signal.

  7. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  8. P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells

    PubMed Central

    Prager, Philipp; Hollborn, Margrit; Steffen, Anja; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18. Conclusion High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote

  9. P2X3 antagonists: novel therapeutics for afferent sensitization and chronic pain.

    PubMed

    Ford, Anthony P

    2012-05-01

    SUMMARY Despite decades of innovation and effort, the pharmaceutical needs of countless patients with chronic pain remain underserved. Effective and safe treatments must clearly come from novel approaches, yet targets and molecules selected hitherto have returned little benefit. Antagonism of P2X3 purinoceptors on pain-conveying nerves is a highly novel approach, and compounds from this class are advancing into patient studies. P2X3 channels are found in C- and Aδ-primary afferent neurons in most tissues, and are strikingly specific to pain detection. P2X3 antagonists block peripheral activation of these fibers via ATP, released from most cells by inflammation, injury, stress and distension, and clearly provide an alternative pharmacological mechanism to attenuate pain signals. P2X3 is also expressed presynaptically at central spinal terminals of afferent neurons, where ATP further sensitizes painful signals en route to the brain. The selectivity of P2X3 expression allows hope of a lower potential for adverse effects in brain, gut and cardiovascular tissues - limiting factors for most analgesics. P2X3 receptor-mediated sensitization has been implicated in rodent models in inflammatory, visceral, neuropathic and cancer pain states, as well as in airways hyper-reactivity, migraine and visceral organ irritability. Although we are often reminded that the effects of new medicines can translate poorly into clinical effectiveness, the broad efficacy seen following P2X3 inhibition in rodent models strengthens the prospect that an unprecedented mechanism to counter sensitization of afferent pathways may offer some merciful relief to millions of patients struggling daily with persistent discomfort and pain.

  10. Characterization of protoberberine analogs employed as novel human P2X{sub 7} receptor antagonists

    SciTech Connect

    Lee, Ga Eun; Lee, Won-Gil; Lee, Song-Yi; Lee, Cho-Rong; Park, Chul-Seung; Chang, Sunghoe; Park, Sung-Gyoo; Song, Mi-Ryoung; Kim, Yong-Chul

    2011-04-15

    The P2X{sub 7} receptor (P2X{sub 7}R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X{sub 7}R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X{sub 7}-expressing HEK293 cells, with IC{sub 50} values of 100-300 nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca{sup 2+} influx were strongly inhibited by compounds 3-5 in patch-clamp and Ca{sup 2+} influx assays. The antagonists also effectively suppressed downstream signaling of P2X{sub 7} receptors including IL-1{beta} release and phosphorylation of ERK1/2 and p38 proteins in hP2X{sub 7}-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X{sub 7} receptor-mediated immune responses by allosteric inhibition of the receptor. - Graphical abstract: Display Omitted

  11. Receptor-mediated mitophagy in yeast and mammalian systems

    PubMed Central

    Liu, Lei; Sakakibara, Kaori; Chen, Quan; Okamoto, Koji

    2014-01-01

    Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease. PMID:24903109

  12. Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration.

    PubMed

    Pajaud, J; Ribault, C; Ben Mosbah, I; Rauch, C; Henderson, C; Bellaud, P; Aninat, C; Loyer, P; Morel, F; Corlu, A

    2015-01-15

    Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration.

  13. Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

    PubMed Central

    Pajaud, J; Ribault, C; Ben Mosbah, I; Rauch, C; Henderson, C; Bellaud, P; Aninat, C; Loyer, P; Morel, F; Corlu, A

    2015-01-01

    Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration. PMID:25590808

  14. P2X4 receptor–eNOS signaling pathway in cardiac myocytes as a novel protective mechanism in heart failure

    PubMed Central

    Yang, Ronghua; Beqiri, Dardan; Shen, Jian-Bing; Redden, John M.; Dodge-Kafka, Kimberly; Jacobson, Kenneth A.; Liang, Bruce T.

    2014-01-01

    We have demonstrated using immunoprecipitation and immunostaining a novel physical association of the P2X4 receptor (P2X4R), a ligand-gated ion channel, with the cardioprotective, calcium-dependent enzyme endothelial nitric oxide synthase (eNOS). Treatment of murine ventricular myocytes with the P2XR agonist 2-methylthioATP (2-meSATP) to induce a current (mainly Na+) increased the formation of nitric oxide (NO), as measured using a fluorescent probe. Possible candidates for downstream effectors mediating eNOS activity include cyclic GMP and PKG or cellular protein nitrosylation. A cardiac-specific P2X4R overexpressing mouse line was protected from heart failure (HF) with improved cardiac function and survival in post-infarct, pressure overload, and calsequestrin (CSQ) overexpression models of HF. Although the role of the P2X4R in other tissues such as the endothelium and monocytes awaits characterization in tissue-specific KO, cardiac-specific activation of eNOS may be more cardioprotective than an increased activity of global systemic eNOS. The intra-myocyte formation of NO may be more advantageous over NO derived externally from a donor. A small molecule drug stimulating this sarcolemmal pathway or gene therapy-mediated overexpression of the P2X4R in cardiac myocytes may represent a new therapy for both ischemic and pressure overloaded HF. PMID:25750695

  15. Receptor-mediated delivery of engineered nucleases for genome modification.

    PubMed

    Chen, Zhong; Jaafar, Lahcen; Agyekum, Davies G; Xiao, Haiyan; Wade, Marlene F; Kumaran, R Ileng; Spector, David L; Bao, Gang; Porteus, Matthew H; Dynan, William S; Meiler, Steffen E

    2013-10-01

    Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.

  16. A(2B) receptors mediate antimitogenesis in vascular smooth muscle cells.

    PubMed

    Dubey, R K; Gillespie, D G; Shue, H; Jackson, E K

    2000-01-01

    Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis

  17. Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons.

    PubMed

    D'Arco, Marianna; Giniatullin, Rashid; Simonetti, Manuela; Fabbro, Alessandra; Nair, Asha; Nistri, Andrea; Fabbretti, Elsa

    2007-08-01

    The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X3 receptor-mediated currents and Ca2+ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X2/3 receptors, consistent with enhanced membrane location of P2X2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X3 subunits and their membrane assembly with P2X2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X3 receptor activity, indicating separate NGF- or CGRP-mediated mechanisms to upregulate P2X3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.

  18. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

    PubMed

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Cho, J Hoon; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.

  19. Potassium 2-(1-hydroxypentyl)-benzoate inhibits ADP-induced rat platelet aggregation through P2Y1-PLC signaling pathways.

    PubMed

    Yang, Hongyan; Xu, Shaofeng; Li, Jiang; Wang, Ling; Wang, Xiaoliang

    2015-09-01

    Potassium 2-(1-hydroxypenty1)-benzoate (dl-PHPB) is a new drug candidate for treatment of ischemic stroke with antiplatelet effect. In this study, we investigated the mechanisms of dl-PHPB in inhibiting platelet aggregation. The ADP-activated P2Y1-Gq-PLC and P2Y12-Gi-AC pathways were observed, respectively. Intravenous injection of dl-PHPB (1.3, 3.9, 12.9 mg/kg) significantly inhibited ADP-, collagen-, and arachidonic acid-induced rat platelet aggregation in a dose-dependent manner, and dl-PHPB had a relatively more potent inhibitory effect on ADP-induced rat platelet aggregation than other agonists. Dl-PHPB also showed a decreased expression of CD62P (a marker for platelet activation) mediated by ADP. Both dl-PHPB and ticlopidine (P2Y12 receptor antagonist) decreased cytoplasmic Ca(2+) concentration. But, dl-PHPB did not reverse the inhibition of PGE1-induced platelet cAMP formation by ADP, which was different from ticlopidine. Further, dl-PHPB instead of ticlopidine showed increasing phospholipase C-β phosphorylation (ser(1105)). The m-3M3FBS, a phospholipase C activator, attenuated the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation and enhanced IP1 accumulation in rat platelets. Dl-PHPB decreased IP1 accumulation induced by ADP but had no effect on IP1 level enhanced by m-3M3FBS. Our results suggest that dl-PHPB has a potent antiplatelet effect, which is mainly through blockade of P2Y1 receptor-PLC-IP3 pathway and decreasing cytoplasmic calcium.

  20. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders

    PubMed Central

    Ford, Anthony P.; Undem, Bradley J.

    2013-01-01

    A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral “hollow” organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists

  1. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders.

    PubMed

    Ford, Anthony P; Undem, Bradley J

    2013-01-01

    A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral "hollow" organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists

  2. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  3. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  4. Hemoglobin uptake by Paracoccidioides spp. is receptor-mediated.

    PubMed

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L; Hernandez, Orville; McEwen, Juan G; Soares, Célia Maria de Almeida

    2014-05-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  5. Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

    PubMed Central

    Xiao, Guangqing

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  6. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice.

    PubMed

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R

    2015-02-01

    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice. PMID:25524412

  7. H-Ras regulation of TRAIL death receptor mediated apoptosis

    PubMed Central

    Chen, Jun-Jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

    2014-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists. PMID:25026275

  8. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  9. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice.

    PubMed

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R

    2015-02-01

    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice.

  10. Sphingosine Phosphate Lyase Regulates Murine Embryonic Stem Cell Proliferation and Pluripotency through an S1P2/STAT3 Signaling Pathway.

    PubMed

    Smith, Gaelen S; Kumar, Ashok; Saba, Julie D

    2013-06-24

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P2 expression. Inhibition of S1P2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.

  11. Bombesin receptor-mediated imaging and cytotoxicity: review and current status

    PubMed Central

    Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

    2010-01-01

    The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

  12. Targeting receptor-mediated endocytotic pathways with nanoparticles: rationale and advances

    PubMed Central

    Xu, Shi; Olenyuk, Bogdan Z.; Okamoto, Curtis T.; Hamm-Alvarez, Sarah F.

    2012-01-01

    Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization. PMID:23026636

  13. Berberine attenuates high glucose-induced fibrosis by activating the G protein-coupled bile acid receptor TGR5 and repressing the S1P2/MAPK signaling pathway in glomerular mesangial cells.

    PubMed

    Yang, Zhiying; Li, Jie; Xiong, Fengxiao; Huang, Junying; Chen, Cheng; Liu, Peiqing; Huang, Heqing

    2016-08-15

    Berberine (BBR) exerts powerful renoprotective effects on diabetic nephropathy (DN), but the underlying mechanisms remain unclear. We previously demonstrated that activation of the G protein-coupled bile acid receptor TGR5 ameliorates diabetic nephropathy by inhibiting the activation of the sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 2 (S1P2) signaling pathway. In this study, we explored the role of TGR5 in the BBR-induced downregulation of sphingosine 1-phosphate receptor 2 (S1P2)/mitogen-activated protein kinase (MAPK)-mediated fibrosis in glomerular mesangial cells (GMCs). Results showed that, BBR suppressed the expression of FN, ICAM-1, and TGF-β1 in high-glucose cultures of GMCs, and the phosphorylation level of c-Jun/c-Fos was downregulated. The high glucose lowered TGR5 expression in a time-dependent manner; this effect was reversed by BBR in a dose-dependent manner. The TGR5 agonist INT-777 decreased the high glucose-induced FN, ICAM-1, and TGF-β1 protein contents. In addition, TGR5 siRNA blocked S1P2 degradation by BBR. And MAPK signaling, which plays important regulatory roles in the pathological progression of DN, was activated by TGR5 siRNA. Apart from this, MAPK signaling as well as FN, ICAM-1, and TGF-β1 suppressed by BBR under high glucose conditions were limited by TGR5 depletion. Thus, BBR decreases FN, ICAM-1, and TGF-β1 levels under high glucose conditions in GMCs possibly by activating TGR5 and inhibiting S1P2/MAPK signaling. PMID:27292312

  14. In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

    PubMed

    Bulley, Simon J; Droubi, Alaa; Clarke, Jonathan H; Anderson, Karen E; Stephens, Len R; Hawkins, Phillip T; Irvine, Robin F

    2016-09-20

    Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal. PMID:27601656

  15. CNS remyelination as a novel reparative approach to neurodegenerative diseases: The roles of purinergic signaling and the P2Y-like receptor GPR17.

    PubMed

    Fumagalli, Marta; Lecca, Davide; Abbracchio, Maria P

    2016-05-01

    Oligodendrocytes are the myelin-forming cells in the CNS. They enwrap axons, thus permitting fast impulse transmission and exerting trophic actions on neurons. Demyelination accompanied by neurological deficit is a rather frequent condition that is not only associated with multiple sclerosis but has been also recognized in several other neurodegenerative diseases, including brain trauma and stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Recently, alterations of myelin function have been also reported in neuropsychiatric diseases, like depression and autism. Highly relevant for therapeutic purposes, oligodendrocyte precursor cells (OPCs) still persist in the adult brain and spinal cord. These cells are normally rather quiescent, but under specific circumstances, they can be stimulated to undergo differentiation and generate mature myelinating oligodendrocytes. Thus, approaches aimed at restoring myelin integrity and at fostering a correct oligodendrocyte function are now viewed as novel therapeutic opportunities for both neurodegenerative and neuropsychiatric diseases. Both OPCs and mature oligodendrocytes express purinergic receptors. For some of these receptors, expression is restricted at specific differentiation stages, suggesting key roles in OPCs maturation and myelination. Some of these receptors are altered under demyelinating conditions, suggesting that their dysregulation may contribute to disease development and could represent adequate new targets for remyelinating therapies. Here, we shall describe the current literature available on all these receptors, with special emphasis on the P2Y-like GPR17 receptor, that represents one of the most studied receptor subtypes in these cells. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26453964

  16. ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade.

    PubMed

    Ehrlich, Lorna S; Medina, Gisselle N; Carter, Carol A

    2011-10-21

    Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P(7)TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY(36)PX(n)L motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that "gates" Ca(2+) release from intracellular stores, triggers Ca(2+) cell influx and thereby functions as a major regulator of Ca(2+) signaling. In the present study, we determined whether the L domain links Gag to Ca(2+) signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca(2+) was elevated, events indicative of induction of Ca(2+) signaling. The results suggest that L domain function, ESCRT machinery and Ca(2+) signaling are linked events in Gag release.

  17. Imaging receptor-mediated endocytosis with a polymeric nanoparticle-based coherent anti-stokes Raman scattering probe.

    PubMed

    Tong, Ling; Lu, Yanhui; Lee, Robert J; Cheng, Ji-Xin

    2007-08-23

    Coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize receptor-mediated endocytosis and intracellular trafficking with the aid of a CARS probe. The probe was made of 200-nm polystyrene particles encapsulated in folate-targeted liposomes. By tuning (omega(p) - omega(s)) to 3045 cm(-1), which corresponds to the aromatic C-H stretching vibration, the polystyrene nanoparticles with a high density of aromatic C-H bonds were detected with a high signal-to-noise ratio, while the epi-detected CARS signal from cellular organelles was cancelled by the destructive interference between the resonant contribution from the aliphatic C-H vibration and the nonresonant contribution. Without any photobleaching, the CARS probe allowed single-particle tracking analysis of intracellular endosome transport. No photodamage to cells was observed under the current experimental conditions. These results show the advantages and potential of using a CARS probe to study cellular processes. PMID:17663581

  18. Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity

    PubMed Central

    Lo, Fu-Sun; Erzurumlu, Reha S.

    2016-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated gene MET tyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAA receptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAA receptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAA receptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. PMID:27030755

  19. The relationship between P2X4 and P2X7: a physiologically important interaction?

    PubMed

    Craigie, Eilidh; Birch, Rebecca E; Unwin, Robert J; Wildman, Scott S

    2013-01-01

    Purinergic signaling within the kidney is becoming an important focus in the study of renal health and disease. The effectors of ATP signaling, the P2Y and P2X receptors, are expressed to varying extents in and along the nephron. There are many studies demonstrating the importance of the P2Y2 receptor on kidney function, and other P2 receptors are now emerging as participants in renal regulation. The P2X4 receptor has been linked to epithelial sodium transport in the nephron and expression levels of the P2X7 receptor are up-regulated in certain pathophysiological states. P2X7 antagonism has been shown to ameliorate rodent models of DOCA salt-induced hypertension and P2X4 null mice are hypertensive. Interestingly, polymorphisms in the genetic loci of P2X4 and P2X7 have been linked to blood pressure variation in human studies. In addition to the increasing evidence linking these two P2X receptors to renal function and health, a number of studies link the two receptors in terms of physical associations between their subunits, demonstrated both in vitro and in vivo. This review will analyze the current literature regarding interactions between P2X4 and P2X7 and assess the potential impact of these with respect to renal function. PMID:23966951

  20. Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death.

    PubMed

    Díaz-Trelles, R; Novelli, A; Vega, J A; Marini, A; Fernández-Sánchez, M T

    2000-10-13

    We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 microM), oxatomide (20 microM), and triprolidine (50 microM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 microM) or the endogenous antioxidant melatonin (200 microM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.

  1. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production.

    PubMed

    Bilal, Mahmood Yousif; Houtman, Jon C D

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

  2. Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase.

    PubMed Central

    Bliska, J B; Black, D S

    1995-01-01

    Suppression of host-cell-mediated immunity is a hallmark feature of Yersinia pseudotuberculosis infection. To better understand this process, the interaction of Y. pseudotuberculosis with macrophages and the effect of the virulence plasmid-encoded Yersinia tyrosine phosphatase (YopH) on the oxidative burst was analyzed in a chemiluminescence assay. An oxidative burst was generated upon infection of macrophages with a plasmid-cured strain of Y. pseudotuberculosis opsonized with immunoglobulin G antibody. Infection with plasmid-containing Y. pseudotuberculosis inhibited the oxidative burst triggered by secondary infection with opsonized bacteria. The tyrosine phosphatase activity of YopH was necessary for this inhibition. These results indicate that YopH inhibits Fc receptor-mediated signal transduction in macrophages in a global fashion. In addition, bacterial protein synthesis was not required for macrophage inhibition, suggesting that YopH export and translocation are controlled at the posttranslational level. PMID:7822039

  3. Angiotensin II attenuates NMDA receptor-mediated neuronal cell death and prevents the associated reduction in Bcl-2 expression.

    PubMed

    Schelman, William R; Andres, Robert; Ferguson, Paul; Orr, Brent; Kang, Evan; Weyhenmeyer, James A

    2004-09-10

    While angiotensin II (Ang II) plays a major role in the regulation of blood pressure, fluid homeostasis and neuroendocrine function, recent studies have also implicated the peptide hormone in cell growth, differentiation and apoptosis. In support of this, we have previously demonstrated that Ang II attenuates N-methyl-D-aspartate (NMDA) receptor signaling [Molec. Brain Res. 48 (1997) 197]. To further examine the modulatory role of Ang II on NMDA receptor function, we investigated the effect of angiotensin receptor (AT) activation on NMDA-mediated cell death and the accompanying decrease in Bcl-2 expression. The viability of differentiated N1E-115 and NG108-15 neuronal cell lines was reduced following exposure to NMDA in a dose-dependent manner. MTT analysis (mitochondrial integrity) revealed a decrease in cell survival of 49.4+/-12.3% in NG108 cells and 79.9+/-6.8% in N1E cells following treatment with 10 mM NMDA for 20 h. Cytotoxicity in N1E cells was inhibited by the noncompetitive NMDA receptor antagonist, MK-801. Further, NMDA receptor-mediated cell death in NG108 cells was attenuated by treatment with Ang II. The Ang II effect was inhibited by both AT1 and AT2 receptor antagonists, losartan and PD123319, respectively, suggesting that both receptor subtypes may play a role in the survival effect of Ang II. Since it has been shown that activation of NMDA receptors alters the expression of Bcl-2 family proteins, Western blot analysis was performed in N1E cells to determine whether Ang II alters the NMDA-induced changes in Bcl-2 expression. A concentration-dependent decrease of intracellular Bcl-2 protein levels was observed following treatment with NMDA, and this reduction was inhibited by MK801. Addition of Ang II suppressed the NMDA receptor-mediated reduction in Bcl-2. The Ang II effect on NMDA-mediated changes in Bcl-2 levels was blocked by PD123319, but was not significantly changed by losartan, suggesting AT2 receptor specificity. Taken together, these

  4. A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

    PubMed

    Fresco, Paula; Oliveira, Jorge M A; Kunc, Filip; Soares, Ana Sofia; Rocha-Pereira, Carolina; Gonçalves, Jorge; Diniz, Carmen

    2007-07-01

    This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G

  5. Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

    PubMed Central

    Sobel, Katrin; Menyhart, Katalin; Killer, Nina; Renault, Bérengère; Bauer, Yasmina; Studer, Rolf; Steiner, Beat; Bolli, Martin H.; Nayler, Oliver; Gatfield, John

    2013-01-01

    Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways. PMID:23589284

  6. Odorant receptor-mediated sperm activation in disease vector mosquitoes

    PubMed Central

    Pitts, R. Jason; Liu, Chao; Zhou, Xiaofan; Malpartida, Juan C.; Zwiebel, Laurence J.

    2014-01-01

    Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution. PMID:24550284

  7. Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells

    PubMed Central

    Zhang, Ping-Ping; Zhang, Gong; Zhou, Wei; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-01-01

    ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca2+]i through the IP3-sensitive pathway and was blocked by intracellular Ca2+-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca2+/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs. PMID:27357477

  8. MFR, a Putative Receptor Mediating the Fusion of Macrophages

    PubMed Central

    Saginario, Charles; Sterling, Hyacinth; Beckers, Cornelius; Kobayashi, Ruji; Solimena, Michele; Ullu, Elisabetta; Vignery, Agnès

    1998-01-01

    We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRPα, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events. PMID:9774638

  9. Nucleus incertus Orexin2 receptors mediate alcohol seeking in rats.

    PubMed

    Kastman, Hanna E; Blasiak, Anna; Walker, Leigh; Siwiec, Marcin; Krstew, Elena V; Gundlach, Andrew L; Lawrence, Andrew J

    2016-11-01

    Alcoholism is a chronic relapsing disorder and a major global health problem. Stress is a key precipitant of relapse in human alcoholics and in animal models of alcohol seeking. The brainstem nucleus incertus (NI) contains a population of relaxin-3 neurons that are highly responsive to psychological stressors; and the ascending NI relaxin-3/RXFP3 signalling system is implicated in stress-induced reinstatement of alcohol seeking. The NI receives orexinergic innervation and expresses orexin1 (OX1) and orexin2 (OX2) receptor mRNA. In alcohol-preferring (iP) rats, we examined the impact of yohimbine-induced reinstatement of alcohol seeking on orexin neuronal activation, and the effect of bilateral injections into NI of the OX1 receptor antagonist, SB-334867 (n = 16) or the OX2 receptor antagonist, TCS-OX2-29 (n = 8) on stress-induced reinstatement of alcohol seeking. We also assessed the effects of orexin-A on NI neuronal activity and the involvement of OX1 and OX2 receptors using whole cell patch-clamp recordings in rat brain slices. Yohimbine-induced reinstatement of alcohol seeking activated orexin neurons. Bilateral NI injections of TCS-OX2-29 attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injection of SB-334867 had no significant effect. In line with these data, orexin-A (600 nM) depolarized a majority of NI neurons recorded in coronal brain slices (18/28 cells), effects prevented by bath application of TCS-OX2-29 (10 μM), but not SB-334867 (10 μM). These data suggest an excitatory orexinergic input to NI contributes to yohimbine-induced reinstatement of alcohol seeking, predominantly via OX2 receptor signalling. PMID:27395787

  10. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  11. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  12. P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

    PubMed Central

    Viering, Daan H. H. M.; Bos, Caro; Bindels, René J. M.; Hoenderop, Joost G. J.

    2016-01-01

    ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg2+ and Na+ reabsorption, but its role in ion transport remains unknown. In this study, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na+, K+, Mg2+ and Ca2+ were not detected between P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions. PMID:27254077

  13. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  14. Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity.

    PubMed

    Yoon, Byung-Il; Hirabayashi, Yoko; Kawasaki, Yasushi; Kodama, Yukio; Kaneko, Toyozo; Kanno, Jun; Kim, Dae-Yong; Fujii-Kuriyama, Yoshiaki; Inoue, Tohru

    2002-11-01

    regulated by AhR signaling.

  15. Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense

    PubMed Central

    Zimmermann, Sabine; Nürnberger, Thorsten; Frachisse, Jean-Marie; Wirtz, Wolfgang; Guern, Jean; Hedrich, Rainer; Scheel, Dierk

    1997-01-01

    Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley. PMID:11038609

  16. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  17. Enhancement of steroid receptor-mediated transcription for the development of highly responsive bioassays.

    PubMed

    Willemsen, Philippe; Scippo, Marie-Louise; Maghuin-Rogister, Guy; Martial, Joseph A; Muller, Marc

    2005-06-01

    We have previously generated several transformed human mammary cell lines for the detection of steroid receptor-mediated activities and used these cell lines to detect and characterize steroid hormone (ant)agonistic compounds. In this report, we describe the specific optimization procedures used to enhance receptor-mediated transcription through the human glucocorticoid, progesterone and androgen receptors, respectively. Sodium arsenite-induced chemical stress leads to a substantial and specific increase in the glucocorticoid receptor-mediated transcription, resulting in maximal stimulations of more than 2000-fold by the agonist dexamethasone. Similarly, a combined treatment with forskolin (an activator of adenylate cyclase) and trichostatin A (an inhibitor of histone deacetylases) leads to a synergistic enhancement of progesterone or androgen stimulation, resulting in a maximal induction of more than 200-fold or about 100-fold, respectively. The enhanced responses to specific steroids are mediated by the corresponding nuclear receptor. We show that by using these enhanced transcriptional stimulation protocols, it is possible to detect lower amounts of steroid hormones without substantially affecting the relative biological activities of various agonists. Finally, the application of these enhanced reporter cell assays to real biological samples from meat-producing animals is evaluated, and some validation parameters are presented.

  18. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  19. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells.

    PubMed

    Wang, Lingwei; Karlsson, Lena; Moses, Sara; Hultgårdh-Nilsson, Anna; Andersson, Maria; Borna, Catharina; Gudbjartsson, Tomas; Jern, Sverker; Erlinge, David

    2002-12-01

    P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

  20. Receptor-mediated endocytosis of polypeptide hormones is a regulated process: inhibition of (125I)iodoinsulin internalization in hypoinsulinemic diabetes of rat and man

    SciTech Connect

    Carpentier, J.L.; Robert, A.; Grunberger, G.; van Obberghen, E.; Freychet, P.; Orci, L.; Gorden, P.

    1986-07-01

    Much data suggest that receptor-mediated endocytosis is regulated in states of hormone excess. Thus, in hyperinsulinemic states there is an accelerated loss of cell surface insulin receptors. In the present experiments we addressed this question in hypoinsulinemic states, in which insulin binding to cell surface receptors is generally increased. In hepatocytes obtained from hypoinsulinemic streptozotocin-induced diabetic rats, (/sup 125/I)iodoglucagon internalization was increased, while at the same time (/sup 125/I)iodoinsulin internalization was decreased. The defect in (/sup 125/I)iodoinsulin internalization was corrected by insulin treatment of the animal. In peripheral blood monocytes from patients with type I insulinopenic diabetes, internalization of (/sup 125/I)iodoinsulin was impaired; this defect was not present in insulin-treated patients. These data in the hypoinsulinemic rat and human diabetes suggest that receptor-mediated endocytosis is regulated in states of insulin deficiency as well as insulin excess. Delayed or reduced internalization of the insulin-receptor complex could amplify the muted signal caused by deficient hormone secretion.

  1. 5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons

    PubMed Central

    Wang, Haitao; Hu, Lingli; Liu, Chunhua; Su, Zhenghui; Wang, Lihui; Pan, Guangjin; Guo, Yiping; He, Jufang

    2016-01-01

    Neural progenitors differentiated from induced pluripotent stem cells (iPS) hold potentials for treating neurological diseases. Serotonin has potent effects on neuronal functions through multiple receptors, underlying a variety of neural disorders. Glutamate and GABA receptors have been proven functional in neurons differentiated from iPS, however, little is known about 5-HT receptor-mediated modulation in such neuronal networks. In the present study, human iPS were differentiated into cells possessing featured physiological properties of cortical neurons. Whole-cell patch-clamp recording was used to examine the involvement of 5-HT2 receptors in functional modulation of GABAergic synaptic transmission. We found that serotonin and DOI (a selective agonist of 5-HT2A/C receptor) reversibly reduced GABA-activated currents, and this 5-HT2A/C receptor mediated inhibition required G protein, PLC, PKC, and Ca2+ signaling. Serotonin increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), which could be mimicked by α-methylserotonin, a 5-HT2 receptor agonist. In contrast, DOI reduced both frequency and amplitude of mIPSCs. These findings suggested that in iPS-derived human neurons serotonin postsynaptically reduced GABAa receptor function through 5-HT2A/C receptors, but presynaptically other 5-HT2 receptors counteracted the action of 5-HT2A/C receptors. Functional expression of serotonin receptors in human iPS-derived neurons provides a pre-requisite for their normal behaviors after grafting. PMID:26837719

  2. Inhibitory effect of ginsenosides on NMDA receptor-mediated signals in rat hippocampal neurons.

    PubMed

    Kim, Sunoh; Ahn, Kwangseog; Oh, Tae Hwan; Nah, Seung-Yeol; Rhim, Hyewhon

    2002-08-16

    Alternative medicines such as herbal products are increasingly being used for preventive and therapeutic purposes. Ginseng is the best known and most popular herbal medicine used worldwide. In spite of some beneficial effects of ginseng on the CNS, little scientific evidence shows at the cellular level. In the present study, we have examined the direct modulation of ginseng on the activation of glutamate, especially NMDA, receptors in cultured hippocampal neurons. Using fura-2-based digital imaging techniques, we found ginseng total saponins inhibited NMDA-induced but less effectively glutamate-induced increase in [Ca2+]i. Ginseng total saponins also modulated Ca2+ transients evoked by depolarization with 50mM KCl along with its own effects on [Ca2+]i. Furthermore, we demonstrated that ginsenoside Rg3 is an active component for ginseng actions on NMDA receptors. The data obtained suggest that the inhibition of NMDA receptors by ginseng, in particular by ginsenoside Rg3, could be one of the mechanisms for ginseng-mediated neuroprotective actions.

  3. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies

    PubMed Central

    2016-01-01

    N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca2+ influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca2+ homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases.

  4. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies

    PubMed Central

    2016-01-01

    N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca2+ influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca2+ homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases. PMID:27630777

  5. Interleukin-21 receptor-mediated signals control autoreactive T cell infiltration in pancreatic islets.

    PubMed

    Van Belle, Tom L; Nierkens, Stefan; Arens, Ramon; von Herrath, Matthias G

    2012-06-29

    It remains unclear how interleukin-21 receptor (IL-21R) contributes to type 1 diabetes. Here we have shown that dendritic cells (DCs) in the pancreas required IL-21R not for antigen uptake, but to acquire the chemokine receptor CCR7 and migrate into the draining lymph node. Consequently, less antigen, major histocompatibility complex (MHC) class II, and CD86 was provided to autoreactive effector cells in Il21r(-/-) mice, impairing CD4(+) T cell activation, CD40:CD40L interactions, and pancreatic infiltration by autoreactive T cells. CD40 crosslinking restored defective CD4(+) cell expansion and CD4 independently expanded autoreactive CD8(+) cells, but CD8(+) cells still required CD4(+) cells to reach the pancreas and induce diabetes. Diabetes induction by transferred T cells required IL-21R-sufficient host antigen-presenting cells. Transferring IL-21R-sufficient DCs broke diabetes resistance in Il21r(-/-) mice. We conclude that IL-21R controls both antigen transport by DCs and the crucial beacon function of CD4(+) cells for autoreactive CD8(+) cells to reach the islets.

  6. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies.

    PubMed

    Carvajal, Francisco J; Mattison, Hayley A; Cerpa, Waldo

    2016-01-01

    N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca(2+) influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca(2+) homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases. PMID:27630777

  7. Activation of the transcription factor FosB/activating protein-1 (AP-1) is a prominent downstream signal of the extracellular nucleotide receptor P2RX7 in monocytic and osteoblastic cells.

    PubMed

    Gavala, Monica L; Hill, Lindsay M; Lenertz, Lisa Y; Karta, Maya R; Bertics, Paul J

    2010-10-29

    Activation of the ionotropic P2RX7 nucleotide receptor by extracellular ATP has been implicated in modulating inflammatory disease progression. Continuous exposure of P2RX7 to ligand can result in apoptosis in many cell types, including monocytic cells, whereas transient activation of P2RX7 is linked to inflammatory mediator production and the promotion of cell growth. Given the rapid hydrolysis of ATP in the circulation and interstitial space, transient activation of P2RX7 appears critically important for its action, yet its effects on gene expression are unclear. The present study demonstrates that short-term stimulation of human and mouse monocytic cells as well as mouse osteoblasts with P2RX7 agonists substantially induces the expression of several activating protein-1 (AP-1) members, particularly FosB. The potent activation of FosB after P2RX7 stimulation is especially noteworthy considering that little is known concerning the role of FosB in immunological regulation. Interestingly, the magnitude of FosB activation induced by P2RX7 stimulation appears greater than that observed with other known inducers of FosB expression. In addition, we have identified a previously unrecognized role for FosB in osteoblasts with respect to nucleotide-induced expression of cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin biosynthesis from arachidonic acid and is critical for osteoblastic differentiation and immune behavior. The present studies are the first to link P2RX7 action to FosB/AP-1 regulation in multiple cell types, including a role in nucleotide-induced COX-2 expression, and support a role for FosB in the control of immune and osteogenic function by P2RX7. PMID:20813842

  8. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  9. A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

    PubMed

    Mishra, Amarjit; Guo, Yujie; Zhang, Li; More, Sunil; Weng, Tingting; Chintagari, Narendranath Reddy; Huang, Chaoqun; Liang, Yurong; Pushparaj, Samuel; Gou, Deming; Breshears, Melanie; Liu, Lin

    2016-10-01

    Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses. PMID:27559050

  10. Receptor-mediated delivery of photoprotective agents by low-density lipoprotein

    SciTech Connect

    Mosley, S.T.; Yang, Y.L.; Falck, J.R.; Anderson, R.G.W.

    1984-12-01

    Low density lipoprotein (LDL) has been used to deliver toxic molecules to cells by receptor-mediated endocytosis. In these studies, the cholesteryl ester core of LDL was replaced with a lipophilic, toxic molecule. The authors report that photoprotective azo dyes can be stably incorporated into LDL, and that this reconstituted LDL protects cells from the photosensitizing action of pyrene methanol (PM) in a receptor-dependent process. The photoprotective action of the azo dye is due to its ability to scavenge singlet oxygen that is produced by the photosensitive agent in response to UV light.

  11. Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted) Tetrahymena pyriformis.

    PubMed

    Kovács, P; Sundermann, C A; Csaba, G

    1996-08-15

    Receptor-mediated endocytosis by Tetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin receptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals and Tetrahymena, and galactose, did not influence the uptake of TRITC-Con A.

  12. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  13. 2p2 Team News

    NASA Astrophysics Data System (ADS)

    Jones, H.

    2000-06-01

    The 2p2 Team continued towards the implementation at the 2.2-m of the same BOB (Broker for Observation Blocks) observing interface as seen at other ESO telescopes. This requires an interface to be written between the existing BOB software and the non-VLT compatible control software for the Wide-Field Imager (WFI) and 2.2-m. Cristian Urrutia, Tatiana Paz and Eduardo Robledo are heading its development. With this software in place, observers can use the VLT Phase 2 Proposal Preparation System (P2PP) for definition of their exposures, whether they are for Visitor or Service Mode.

  14. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  15. CD44, α4 integrin, and fucoidin receptor-mediated phagocytosis of apoptotic leukocytes

    PubMed Central

    Johnson, Jacob D.; Hess, Krista L.; Cook-Mills, Joan M.

    2011-01-01

    Various types of phagocytes mediate the clearance of apoptotic cells. We previously reported that human and murine high endothelial venule (HEV) cells ingest apoptotic cells. In this report, we examined endothelial cell fucoidin receptor-mediated phagocytosis using a murine endothelial cell model mHEV. mHEV cell recognition of apoptotic leukocytes was blocked by fucoidin but not by other phagocytic receptor inhibitors such as mannose, fucose, N-acetylglucosamine, phosphatidylserine (PS), or blocking anti-PS receptor antibodies. Thus, the mHEV cells used fucoidin receptors for recognition and phagocytosis of apoptotic leukocytes. The fucoidin receptor-mediated endothelial cell phagocytosis was specific for apoptotic leukocytes, as necrotic cells were not ingested. This is in contrast to macrophages, which ingest apoptotic and necrotic cells. Endothelial cell phagocytosis of apoptotic cells did not alter viable lymphocyte migration across these endothelial cells. Antibody blocking of CD44 and α4 integrin on the apoptotic leukocyte inhibited this endothelial cell phagocytosis, suggesting a novel function for these adhesion molecules in the removal of apoptotic targets. The removal of apoptotic leukocytes by endothelial cells may protect the microvasculature, thus ensuring that viable lymphocytes can successfully migrate into tissues. PMID:12960273

  16. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis

    PubMed Central

    Richards, David M.; Endres, Robert G.

    2016-01-01

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery. PMID:27185939

  17. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  18. Melanocortin MC(4) receptor-mediated feeding and grooming in rodents.

    PubMed

    Mul, Joram D; Spruijt, Berry M; Brakkee, Jan H; Adan, Roger A H

    2013-11-01

    Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced.

  19. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  20. Role of caveolin 1 in AT1a receptor-mediated uptake of angiotensin II in the proximal tubule of the kidney

    PubMed Central

    Li, Xiao C.; Gu, Victor; Miguel-Qin, Elise

    2014-01-01

    Caveolin 1 (CAV-1) functions not only as a constitutive scaffolding protein of caveolae but also as a vesicular transporter and signaling regulator. In the present study, we tested the hypothesis that CAV-1 knockout (CAV-1 KO) inhibits ANG II type 1 [AT1 (AT1a)] receptor-mediated uptake of ANG II in the proximal tubule and attenuates blood pressure responses in ANG II-induced hypertension. To determine the role of CAV-1 in mediating the uptake of FITC-labeled ANG II, wild-type (WT) mouse proximal convoluted tubule cells were transfected with CAV-1 small interfering (si)RNA for 48 h before AT1 receptor-mediated uptake of FITC-labeled ANG II was studied. CAV-1 siRNA knocked down CAV-1 expression by >90% (P < 0.01) and inhibited FITC-labeled ANG II uptake by >50% (P < 0.01). Moreover, CAV-1 siRNA attenuated ANG II-induced activation of MAPK ERK1/2 and Na+/H+ exchanger 3 expression, respectively (P < 0.01). To determine whether CAV-1 regulates ANG II uptake in the proximal tubule, Alexa 488-labeled ANG II was infused into anesthetized WT and CAV-1 KO mice for 60 min (20 ng/min iv). Imaging analysis revealed that Alexa 488-labeled ANG II uptake was decreased by >50% in CAV-1 KO mice (P < 0.01). Furthermore, Val5-ANG II was infused into WT and CAV-1 KO mice for 2 wk (1.5 mg·kg−1·day−1 ip). Basal systolic pressure was higher, whereas blood pressure and renal excretory and signaling responses to ANG II were attenuated, in CAV-1 KO mice (P < 0.01). We concluded that CAV-1 plays an important role in AT1 receptor-mediated uptake of ANG II in the proximal tubule and modulates blood pressure and renal responses to ANG II. PMID:25164083

  1. Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 CellsV⃞

    PubMed Central

    Morelli, Anna; Chiozzi, Paola; Chiesa, Anna; Ferrari, Davide; Sanz, Juana M.; Falzoni, Simonetta; Pinton, Paolo; Rizzuto, Rosario; Olson, Michael F.; Di Virgilio, Francesco

    2003-01-01

    The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I. PMID:12857854

  2. Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

    PubMed Central

    Hogg, David W; Pamenter, Matthew E; Dukoff, David J; Buck, Leslie T

    2015-01-01

    Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited

  3. Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans

    PubMed Central

    Moura, GEDD; Lucena, SV; Lima, MA; Nascimento, FD; Gesteira, TF; Nader, HB; Paredes-Gamero, EJ; Tersariol, ILS

    2015-01-01

    Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation. PMID:27551441

  4. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  5. An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

    PubMed

    Rizk, Shahir S; Luchniak, Anna; Uysal, Serdar; Brawley, Crista M; Rock, Ronald S; Kossiakoff, Anthony A

    2009-07-01

    We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.

  6. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  7. Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts

    SciTech Connect

    Agmon, V.; Dinur, T.; Cherbu, S.; Dagan, A.; Gatt, S. )

    1991-10-01

    Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

  8. Receptor mediated uptake of paclitaxel from a synthetic high density lipoprotein nanocarrier.

    PubMed

    Mooberry, Linda K; Nair, Maya; Paranjape, Sulabha; McConathy, Walter J; Lacko, Andras G

    2010-01-01

    The purpose of these studies was to determine the mechanism(s) whereby paclitaxel (PTX), is taken up by cancer cells, once encapsulated into synthetic/reconstituted high density lipoprotein (rHDL). The uptake of PTX was found to be facilitated by the scavenger receptor type B-1 (SR-B1) when drug-loaded rHDL particles were incubated with cells that express the SRB1 receptor. Studies with double-labeled, PTX containing rHDL nanoparticles showed that prostate cancer (PC-3) cells incorporated PTX primarily via a selective (SR-B1 type) uptake mechanism. In the presence of a 10-fold excess of plasma HDL, PTX uptake decreased to 30% of the control. These findings suggest that the incorporation of lipophilic drugs by cancer cells from rHDL nanoparticles is facilitated by a receptor mediated (SR-B1) mechanism.

  9. Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier

    PubMed Central

    Lajoie, Jason M.; Shusta, Eric V.

    2016-01-01

    Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer’s disease, stroke and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence, therapeutic potential, of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT targeting strategies have been developed over the past 20 years, and this review will explore exciting recent advances, with a particular emphasis on those studies showing brain targeting in vivo. PMID:25340933

  10. Effects of 2-phenoxyethanol on N-methyl-D-aspartate (NMDA) receptor-mediated ion currents.

    PubMed

    Musshoff, U; Madeja, M; Binding, N; Witting, U; Speckmann, E J

    1999-02-01

    The actions were examined of 17 frequently used glycol ether compounds on the glutamate receptor-mediated ion currents. The receptors were expressed in Xenopus oocytes by injection of rat brain mRNA. Most of the 17 glycol ethers exerted no effects on the glutamate subreceptors activated by kainate and N-methyl-D-aspartate (NMDA), whereas 2-phenoxyethanol (ethylene glycol monophenyl ether) caused a considerable reduction of NMDA-induced membrane currents in a reversible and concentration-dependent manner. The threshold concentration of the ethylene glycol monophenyl ether effect was < 10 mumol/l. The concentration for a 50% inhibition (IC50) was approximately 360 mumol/l. The results indicate a neurotoxic potential for 2-phenoxyethanol.

  11. 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.

  12. AMPA receptor-mediated miniature synaptic calcium transients in GluR2 null mice.

    PubMed

    Wang, Sabrina; Jia, Zhengping; Roder, John; Murphy, Timothy H

    2002-07-01

    AMPA-type glutamate receptors are normally Ca(2+) impermeable due to the expression of the GluR2 receptor subunit. By using GluR2 null mice we were able to detect miniature synaptic Ca(2+) transients (MSCTs) associated with AMPA-type receptor-mediated miniature synaptic currents at single synapses in primary cortical cultures. MSCTs and associated Ca(2+) transients were monitored under conditions that isolated responses mediated by AMPA or N-methyl-D-aspartate (NMDA) receptors. As expected, addition of the antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 3 microM) blocked the AMPA receptor-mediated MSCTs. Voltage-gated Ca(2+) channels did not contribute to AMPA MSCTs because CdCl(2) (0.1-0.2 mM) did not significantly alter the frequency or the amplitude of the MSCTs. The amplitude of AMPA MSCTs appeared to be regulated independently from event frequency since the two measures were not correlated (R = 0.023). Synapses were identified that only expressed MSCTs attributed to either NMDA or AMPA receptors. At synapses with only NMDA responses, MSCT amplitude was significantly lower (by 40%) than synapses expressing both NMDA and AMPA responses. At synapses that showed MSCTs mediated by both AMPA and NMDA receptors, the amplitude of the transients in each condition was positively correlated (R = 0.94). Our results suggest that when AMPA and NMDA receptors are co-expressed at synapses, mechanisms exist to ensure proportional scaling of each receptor type that are distinct from the presynaptic factors controlling the frequency of miniature release. PMID:12091530

  13. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    PubMed

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

  14. Glutamate receptor-mediated oligodendrocyte toxicity in periventricular leukomalacia: a protective role for topiramate.

    PubMed

    Follett, Pamela L; Deng, Wenbin; Dai, Weimin; Talos, Delia M; Massillon, Leon J; Rosenberg, Paul A; Volpe, Joseph J; Jensen, Frances E

    2004-05-01

    Periventricular leukomalacia is a form of hypoxic-ischemic cerebral white matter injury seen most commonly in premature infants and is the major antecedent of cerebral palsy. Glutamate receptor-mediated excitotoxicity is a predominant mechanism of hypoxic-ischemic injury to developing cerebral white matter. We have demonstrated previously the protective effect of AMPA-kainate-type glutamate receptor blockade in a rodent model of periventricular leukomalacia. The present study explores the therapeutic potential of glutamate receptor blockade for hypoxic-ischemic white matter injury. We demonstrate that AMPA receptors are expressed on developing human oligodendrocytes that populate fetal white matter at 23-32 weeks gestation, the period of highest risk for periventricular leukomalacia. We show that the clinically available anticonvulsant topiramate, when administered post-insult in vivo, is protective against selective hypoxic-ischemic white matter injury and decreases the subsequent neuromotor deficits. We further demonstrate that topiramate attenuates AMPA-kainate receptor-mediated cell death and calcium influx, as well as kainate-evoked currents in developing oligodendrocytes, similar to the AMPA-kainate receptor antagonist 6-nitro-7-sulfamoylbenzo-(f)quinoxaline-2,3-dione (NBQX). Notably, protective doses of NBQX and topiramate do not affect normal maturation and proliferation of oligodendrocytes either in vivo or in vitro. Taken together, these results suggest that AMPA-kainate receptor blockade may have potential for translation as a therapeutic strategy for periventricular leukomalacia and that the mechanism of protective efficacy of topiramate is caused at least in part by attenuation of excitotoxic injury to premyelinating oligodendrocytes in developing white matter.

  15. Relationship between Ah receptor-mediated polychlorinated biphenyl (PCB)-induced humoral immunosuppression and thymic atrophy.

    PubMed

    Silkworth, J B; Antrim, L

    1985-12-01

    Thymic atrophy and humoral immunosuppression by certain polychlorinated biphenyls is associated with the aromatic hydrocarbon (Ah) receptor in mice. We examined the relationship between these two toxic effects. 3,3',4,4'-Tetrachlorobiphenyl (TCB), which causes immunosuppression and thymic atrophy, and 2,3,3',4,4',5-hexachlorobiphenyl, which causes immunosuppression without thymic atrophy, were administered i.p. to C57BL/6 mice at 0, 35 and 350 mumol/kg b.wt. 2 days before i.v. immunization with 10 micrograms of Escherichia coli lipopolysaccharide. Both congeners caused significant suppression of the day 4 anti-lipopolysaccharide plaque-forming cell response/spleen (less than or equal to 46% of control). TCB (350 mumol/kg) was also administered 2 days before either a primary or secondary i.p. immunization with sheep erythrocytes. TCB treatment before primary immunization had no effects on the day 5 secondary response, whereas treatment before the secondary immunization significantly inhibited both day 5 immunoglobulin M and immunoglobulin G plaque-forming cells (less than 10 and less than 2% of control, respectively) and decreased serum antibody. TCB administered either 8 or 2 days before or 2 or 4 days after immunization with sheep erythrocytes demonstrated that significant suppression of both plaque-forming cells and serum antibody could occur without thymic atrophy. Immunity was most impaired when TCB was given 2 days before immunization. These results demonstrate that thymic atrophy does not always accompany the severe immunosuppression caused by Ah receptor ligands and suggests that it may not be a sensitive measure of Ah receptor-mediated immunosuppression. The data also suggests that differentiation of B lymphocytes into antibody producing cells is impaired during Ah receptor-mediated gene activation.

  16. Modeling receptor-mediated processes with dioxin: Implications for pharmacokinetics and risk assessment

    SciTech Connect

    Anderson, M.E.; Mills, J.J.; Gargas, M.L. ); Keddersi, L. ); Birnbaum, L.S. ); Neubert, D. ); Greenlee, W.F. )

    1993-02-01

    Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a widespread aromatic hydrocarbon, caused tumors in the liver and other sites when administered chronically to rats at doses as low as 0.01 [mu]g/kg/day. It functions in combination with a cellular protein, the Ah receptor, to alter gene regulation, and this resulting modulation of gene expression is believed to be obligatory for both dioxin toxicity and carcinogenicity. The U.S. EPA is reevaluating its dioxin risk assessment and, as part of this process, will be developing risk assessment approaches for chemicals, such as dioxin, whose toxicity is receptor-mediated. This paper describes a receptor-mediated physiologically based pharmacokinetic (PB-PK) model for the tissue distribution and enzyme-inducing properties of dioxin and discusses the potential role of these models in a biologically motivated risk assessment. In this model, ternary interactions among the Ah receptor, dioxin, and DNA binding sites lead to enhance production of specific hepatic proteins. This model was used to examine the tissue disposition of dioxin and the induction of both a dioxin-binding protein (presumably, cytochrome P4501A2), and cytochrome P4501A1. Tumor promotion correlated more closely with predicted induction of P4501A1 than with induction of hepatic binding proteins. Although increased induction of these proteins is not expected to be causally related to tumor formation, these physiological dosimetry and gene-induction response models will be important for biologically motivated dioxin risk assessments in determining both target tissue dose of dioxin and gene products and in examining the relationship between these gene products and the cellular events more directly involved in tumor promotion.

  17. Fluid Shear Stress Sensitizes Cancer Cells to Receptor-Mediated Apoptosis via Trimeric Death Receptors

    PubMed Central

    Mitchell, Michael J.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer (NK) cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to receptor-mediated apoptosis has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlated with the application of fluid shear stress, and TRAIL-induced apoptosis increased in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis was not affected by the application fluid shear stress. Interestingly, fluid shear stress did not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments revealed that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear force can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents. PMID:25110459

  18. Curcumin Represses NLRP3 Inflammasome Activation via TLR4/MyD88/NF-κB and P2X7R Signaling in PMA-Induced Macrophages

    PubMed Central

    Kong, Fanqi; Ye, Bozhi; Cao, Jiatian; Cai, Xueli; Lin, Lu; Huang, Shanjun; Huang, Weijian; Huang, Zhouqing

    2016-01-01

    Aims: In the NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) inflammasome is closely related to the progression of atherosclerosis. This study aimed to assess the effects of curcumin on NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages and explore its underlying mechanism. Methods: Human monocytic THP-1 cells were pretreated with curcumin for 1 h and subsequently induced with PMA for 48 h. Total protein was collected for Western blot analysis. Cytokine interleukin (IL)-1β release and nuclear factor kappa B (NF-κB) p65 translocation were detected by ELISA assay and cellular NF-κB translocation kit, respectively. Results: Curcumin significantly reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion in PMA-induced macrophages. Moreover, Bay (a NF-κB inhibitor) treatment considerably suppressed the expression of NLRP3 inflammasome in PMA-induced THP-1 cells. Curcumin also markedly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκB-α, and activation of NF-κB in PMA-induced macrophages. In addition, purinergic 2X7 receptor (P2X7R) siRNA was administered, and it significantly decreased NLRP3 inflammasome expression in PMA-induced macrophages. Furthermore, curcumin reversed PMA-stimulated P2X7R activation, which further reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion. Silencing of P2X7R using siRNA also suppressed the activation of NF-κB pathway in PMA-induced macrophages, but P2X7R-silenced cells did not significantly decrease the expression of TLR4 and MyD88. Conclusion: Curcumin inhibited NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB and P2X7R pathways in PMA-induced macrophages. PMID:27777559

  19. P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

    PubMed Central

    Hockley, James R. F.; Tranter, Michael M.; McGuire, Cian; Boundouki, George; Cibert-Goton, Vincent; Thaha, Mohamed A.; Blackshaw, L. Ashley; Michael, Gregory J.; Baker, Mark D.; Knowles, Charles H.; Winchester, Wendy J.

    2016-01-01

    pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease. PMID:26911685

  20. The SLAC P2 Marx

    SciTech Connect

    Kemp, Mark; Benwell, Andrew; Burkhart, Craig; MacNair, David; Nguyen1, Minh; /SLAC

    2012-07-05

    A proposed high energy physics accelerator, the International Linear Collider, will require greater than five hundred rf stations. Each station is composed of a klystron driven by a modulator. Recently, the SLAC P2 Marx was designated the baseline modulator for the ILC. This paper describes some key features of this modulator and presents recent experimental results. The P2 Marx is presently being transported to another facility for lifetime testing. Here, we will gain understanding of how the Marx performs into a klystron load and gain experience operating the Marx for longer periods. Long term plans include the possibility of using this rf station for L-band technology demonstration at SLAC. While the Marx was designed with the ILC in mind, the topology can be readily applied to several different applications. We are currently evaluating the use of the topology for ESS, CLIC, and upgrades for systems at Fermi National Accelerator Laboratory. Because of the modular nature of the cell and the robustness of the control system, many different combinations of series and parallel operation are possible along with different load currents and pulse shapes.

  1. Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells.

    PubMed Central

    Mittman, S; Taylor, W R; Copenhagen, D R

    1990-01-01

    1. Cells in the ganglion cell layer of salamander retinal slices were voltage clamped using patch pipettes. Light elicited transient excitatory postsynaptic currents (EPSCs) in on-off ganglion cells and sustained EPSCs in on ganglion cells. Light-evoked inhibitory postsynaptic currents in these cells could be blocked by 100 microM-bicuculline methobromide and 500 nM-strychnine. 2. In the presence of external Cd2+, at a concentration that blocked light-evoked synaptic inputs, N-methyl-D-aspartate (NMDA) and the non-NMDA-receptor agonists, quisqualate and kainate, gated conductances in both on-off and on ganglion cells. The current-voltage (I-V) curve for the conductance elicited by NMDA had a negative slope between -40 and -70 mV and a reversal potential near 0 mV. The I-V curves for the non-NMDA-receptor-mediated conductances were nearly linear and also had reversal potentials near 0 mV. 3. I-V curves were measured at an early time point near the peak of transient EPSCs and at a later time point during the decay phase of the responses. The late I-V curve had a negative slope below -40 mV. The early I-V curve had a positive slope over the entire voltage range but the slope was greater at positive than at negative potentials. The evoked current reversed near 0 mV at both time points. 4. The region of negative slope of the late I-V curve was eliminated when Mg2+ was removed from the external saline. A slowly decaying component of transient EPSCs was eliminated in 20 microM-DL-2-amino-7-phosphonoheptanoate (AP7), an NMDA-receptor antagonist. 5. Application of 1 microM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA-receptor antagonist at this concentration, blocked a fast component of transient EPSCs. 6. Our results demonstrate that the synaptic inputs to on-off ganglion cells have two components: a slower NMDA-receptor-mediated component having a time-to-peak of 110 +/- 45 ms and an e-fold decay time of 209 +/- 35 ms at -31 mV (mean +/- S.D., n = 5), and a

  2. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  3. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    PubMed

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  4. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  5. Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

    PubMed

    Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio; Falconi, Mattia; Franch, Oskar; Knudsen, Birgitta R; Desideri, Alessandro; Biocca, Silvia

    2016-06-28

    DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells. PMID:27214742

  6. Coated vesicles participate in the receptor-mediated endocytosis of insulin

    PubMed Central

    1983-01-01

    We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross- linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin. PMID:6131074

  7. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  8. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    SciTech Connect

    Majumdar, S.; Basu, S.K. )

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections.

  9. Peptides in Receptor-Mediated Radiotherapy: From Design to the Clinical Application in Cancers

    PubMed Central

    Lozza, Catherine; Navarro-Teulon, Isabelle; Pèlegrin, André; Pouget, Jean-Pierre; Vivès, Eric

    2013-01-01

    Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms. PMID:24093086

  10. Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

    PubMed Central

    Seliverstova, E.V.

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  11. Modulation of receptor-mediated gonadotropin action in rat testes by dietary fat.

    PubMed

    Sebokova, E; Garg, M L; Clandinin, M T

    1988-06-01

    The effect of feeding diets enriched with 18:2 omega 6, 18:3 omega 3, or saturated fatty acids on lipid composition and receptor-mediated action of luteinizing hormone/human chorionic gonadotropin (LH/hCG) in rat testicular plasma membranes was investigated. Linoleic and alpha-linolenic acid treatments reduced total phospholipid and cholesterol content of the testicular plasma membrane and altered membrane phospholipid composition. Change in phospholipid and cholesterol content after feeding the polyunsaturated fats decreased cholesterol to phospholipid ratios and binding capacity of the LH/hCG receptor in the testicular plasma membrane. LH-stimulated adenylate cyclase activity was decreased in animals fed the linolenic acid-rich diet. NaF-stimulated adenylate cyclase activity was decreased in animals fed diets high in either polyunsaturated fatty acid. Decreased plasma membrane LH/hCG receptor content was associated with decreased testosterone production in Leydig cells in response to LH in the linolenic acid-fed group. It is suggested that change in cholesterol-to-phospholipid ratios alters the physical properties of testicular plasma membranes in a manner that influences accessibility of LH/hCG receptors in testicular tissue. PMID:2897795

  12. Characterization of NPY receptors mediating contraction in rat intramyocardial coronary arteries.

    PubMed

    Prieto, D; García-Sacristán, A; Simonsen, U

    1998-09-25

    In vitro experiments in a microvascular myograph were designed in order to characterize the receptor subtypes and the mechanisms underlying the contractions induced by neuropeptide Y (NPY) in rat coronary small arteries. The rank order of potency for NPY-receptor agonist-induced increases in tension in endothelium-intact preparations was polypeptide Y (PYY)> NPY > or = [Leu31Pro34]NPY, while NPY(13-36) only induced small contractions at the highest concentration applied. The selective neuropeptide Y1 receptor antagonist, BIBP 3226, caused rightward shifts in the concentration-response curves for NPY and the slope of the Schild plot was not significantly different from unity. The pA2 value for BIBP 3226 against NPY was 7.88+/-0.15 (n = 6). We have earlier shown that endothelial cell removal does not change the contractile responses induced by NPY, but indomethacin (3 x 10(-6) M) significantly reduced the contractions induced by the peptide. In contrast, the thromboxane receptor antagonist, SQ29548, which abolished the contractions induced by the thromboxane analogue, U46619, did not change the concentration-response curves for NPY. In conclusion, the present study suggests that Y1 receptors mediate NPY-induced contractions in rat coronary resistance arteries, and that a non-thromboxane prostanoid is involved in the contractile mechanism.

  13. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  14. NK-1 receptor mediation of neurogenic plasma extravasation in rat skin.

    PubMed Central

    Andrews, P. V.; Helme, R. D.; Thomas, K. L.

    1989-01-01

    1. Plasma extravasation was induced by electrical nerve stimulation and by perfusion of tachykinins over a vacuum-induced blister base on rat footpad. 2. Stimulation of the sciatic nerve (18 V, 15 Hz, 0.5 ms) for 20 min produced a significant increase in the protein content of the perfusate. The response in capsaicin pretreated rats was only 4% of the control response. This indicates that the electrically-induced plasma extravasation response was mediated by capsaicin-sensitive sensory fibres. 3. Exogenous perfusion of the mammalian tachykinins substance P, neurokinin A and neurokinin B and the non-mammalian tachykinins physalaemin, kassinin and eledoisin was used to determine the tachykinin receptor type mediating the plasma extravasation response. Dose-response curves of the tachykinins (10(-9) M-10(-4) M) gave a rank order of potency of substance P = physalaemin greater than eledoisin greater than or equal to kassinin greater than neurokinin B = neurokinin A. 4. In addition, specific agonists of neurokinin receptors were perfused. Perfusion of [Glp6, D-Pro9] SP6-11 and [Glp6, L-Pro9]SP6-11 demonstrated that the L-Pro isomer was much more potent than the D-Pro isomer. 5. The rank order of potency and the greater potency of [Glp6, L-Pro9]SP6-11 over its D-isomer indicate an NK-1 neurokinin receptor mediates plasma extravasation in rat footpad skin. PMID:2477105

  15. Dynamics of Cytoskeletal Proteins during Fcγ Receptor-mediated Phagocytosis in MacrophagesV⃞

    PubMed Central

    Diakonova, Maria; Bokoch, Gary; Swanson, Joel A.

    2002-01-01

    Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcγ receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37°C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure. PMID:11854399

  16. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  17. Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

    PubMed

    Bayliss, D A; Millhorn, D E; Gallman, E A; Cidlowski, J A

    1987-11-01

    We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration. PMID:3478727

  18. Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

    PubMed

    Pron, G; Mahrour, N; Orlowski, S; Tounekti, O; Poddevin, B; Belehradek, J; Mir, L M

    1999-01-01

    Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

  19. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  20. Structural Basis for Receptor-Mediated Selective Autophagy of Aminopeptidase I Aggregates.

    PubMed

    Yamasaki, Akinori; Watanabe, Yasunori; Adachi, Wakana; Suzuki, Kuninori; Matoba, Kazuaki; Kirisako, Hiromi; Kumeta, Hiroyuki; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Inagaki, Fuyuhiko; Noda, Nobuo N

    2016-06-28

    Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity. PMID:27320913

  1. Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core.

    PubMed Central

    Glössl, J; Schubert-Prinz, R; Gregory, J D; Damle, S P; von Figura, K; Kresse, H

    1983-01-01

    Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues. Images Fig. 2. PMID:6316923

  2. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  3. Receptor-mediated adhesion phenomena. Model studies with the Radical-Flow Detachment Assay.

    PubMed

    Cozens-Roberts, C; Quinn, J A; Lauffenberger, D A

    1990-07-01

    Receptor-mediated cell adhesion phenomena play a vital role in many physiological and biotechnology-related processes. To investigate the physical and chemical factors that influence the cell/surface interaction, we have used a radial flow device, a so-called Radial-Flow Detachment Assay (RFDA). The RFDA allows us to make direct observations of the detachment process under specified experimental conditions. In results reported here, we have studied the detachment of receptor-coated latex beads (prototype cells) from ligand-coated glass surfaces. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine several aspects of the adhesion process with particles having uniform properties that can be varied systematically. Advantages of the RFDA are many, especially direct observation of cell detachment over a range of shear stresses with quantitative measurement of the adhesive force. We focus our studies on the effects of ligand and receptor densities, along with the influence of pH and ionic strength of the medium. These data are analyzed with a mathematical model based on the theoretical framework of Bell, G. I. (1978. Science [Wash. DC]. 200:618-627) and Hammer, D. A. and D. A. Lauffenburger (1987. Biophys. J. 52:475-487). We demonstrate experimental validation of a theoretical expression for the critical shear stress for particle detachment, and show that it is consistent with reasonable estimates for the receptor-ligand bond affinity.

  4. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  5. Receptor and non-receptor mediated formation of superoxide anion and hydrogen peroxide in neutrophils of intensive care patients.

    PubMed

    Manhart, N; Oismüller, C; Lassnig, A; Spittler, A; Sautner, T; Götzinger, P; Függer, R; Roth, E

    1998-11-27

    Generation of reactive oxygen intermediates (ROI) has been implicated in tissue damage in a variety of disease states including sepsis and trauma. On the other hand, generation of ROI in polymorphonuclear granulocytes (PMN) presents a crucial element in the defence of the host against invading microorganisms. In the present study we investigated the generation of superoxide anions (O2-) and hydrogen peroxide (H2O2) by neutrophils (PMN)5 of 17 critically ill patients treated at a intensive care unit (ICU) after polytrauma (n = 6), heart operation (n = 6) or during septic shock (n = 5) using flow cytometry. O2- production of PMN from ICU patients was significantly lower (p < 0.01) than that in healthy volunteers (HV) during non-receptor mediated stimulation with phorbol-myristate-acetate (PMA) but higher (p < 0.001) during receptor mediated stimulation with formylmethionine-leucine-phenylalanine (FMLP). H2O2 generation of PMN from ICU patients was increased after stimulation with FMLP (p < 0.01) and remained unchanged after stimulation with PMA. Patients in septic shock had lower O2(-)-generation of PMN than did injured patients and patients after heart operations. We conclude that receptor mediated formation of O2- and H2O2 is stimulated in ICU patients. However, in patients in septic shock O2(-)-generation decreases, which potentially might contribute to the immunoparalysis present in septic shock.

  6. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  7. Receptor-mediated uptake of ferritin-bound iron by human intestinal Caco-2 cells.

    PubMed

    Kalgaonkar, Swati; Lönnerdal, Bo

    2009-04-01

    Ferritin (Ft) is a large iron (Fe)-binding protein ( approximately 450 kDa) that is found in plant and animal cells and can sequester up to 4500 Fe atoms per Ft molecule. Our previous studies on intestinal Caco-2 cells have shown that dietary factors affect the uptake of Fe from Ft in a manner different from that of Fe from FeSO4, suggesting a different mechanism for cellular uptake. The objective of this study was to determine the mechanism for Ft-Fe uptake using Caco-2 cells. Binding of (59)Fe-labeled Ft at 4 degrees C showed saturable kinetics, and Scatchard analysis resulted in a K(d) of 1.6 muM, strongly indicating a receptor-mediated process. Competitive binding studies with excess unlabelled Ft significantly reduced binding, and uptake studies at 37 degrees C showed saturation after 4 h. Enhancing and blocking endocytosis using Mas-7 (a G-protein activator) and hypertonic medium (0.5 M sucrose), respectively, demonstrated that Ft-Fe uptake by Mas-7-treated cells was 140% of control cells, whereas sucrose treatment resulted in a statistically significant reduction in Ft-Fe uptake by 70% as compared to controls. Inhibition of macropinocytosis with 5-(N,N-dimethyl)-amiloride (Na+/H+ antiport blocker) resulted in a decrease (by approximately 20%) in Ft-Fe uptake at high concentrations of Ft, suggesting that enterocytes can use more than one Ft uptake mechanism in a concentration-dependent manner. These results suggest that Ft uptake by enterocytes is carried out via endocytosis when Ft levels are within a physiological range, whereas Ft at higher concentrations may be absorbed using the additional mechanism of macropinocytosis. PMID:18602806

  8. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  9. Receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid

    SciTech Connect

    Walsh, R.J.; Slaby, F.J.; Posner, B.I.

    1987-05-01

    Prolactin (PRL) interacts with areas of the central nervous system which reside behind the blood-brain barrier. While vascular PRL does not cross this barrier, it is readily accessible to the cerebrospinal fluid (CSF) from which it may gain access to the PRL-responsive areas of the brain. Studies were undertaken to characterize the mechanism responsible for the translocation of PRL from blood to CSF. Rats were given external jugular vein injections of (/sup 125/-I)iodo-PRL in the presence or absence of an excess of unlabeled ovine PRL (oPRL), human GH, bovine GH, or porcine insulin. CSF and choroid plexus were removed 60 min later. CSF samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide slab gels and resultant autoradiographs were analyzed with quantitative microdensitometry. The data revealed that unlabeled lactogenic hormones, viz. oPRL and human GH, caused a statistically significant inhibition of (/sup 125/I)iodo-PRL transport from blood to CSF. In contrast, nonlactogenic hormones, viz bovine GH and insulin, had no effect on (/sup 125/I)iodo-PRL transport into the CSF. An identical pattern of competition was observed in the binding of hormone to the choroid plexus. Furthermore, vascular injections of (/sup 125/I)iodo-PRL administered with a range of concentrations of unlabeled oPRL revealed a dose-response inhibition in the transport of (/sup 125/I)iodo-PRL from blood to CSF. The study demonstrates that PRL enters the CSF by a specific, PRL receptor-mediated transport mechanism. The data is consistent with the hypothesis that the transport mechanism resides at the choroid plexus. The existence of this transport mechanism reflects the importance of the cerebroventricular system in PRL-brain interactions.

  10. Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

    PubMed Central

    Limberg, Jacqueline K.; Peltonen, Garrett L.; Johansson, Rebecca E.; Harrell, John W.; Kellawan, Jeremy M.; Eldridge, Marlowe W.; Sebranek, Joshua J.; Walker, Benjamin J.; Schrage, William G.

    2016-01-01

    Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control. PMID:27375493

  11. Central beta-adrenergic receptors mediate renal nerve activity during stress in conscious spontaneously hypertensive rats.

    PubMed

    Koepke, J P; DiBona, G F

    1985-01-01

    The effects of intracerebroventricular (i.c.v.) administration of beta-adrenergic receptor antagonists (d,l-propranolol or timolol, 30 micrograms in 2 microL of isotonic saline) on the increased renal sympathetic nerve activity and decreased urinary sodium excretion (UNaV) responses to stressful environmental stimulation (air jet to head) in conscious spontaneously hypertensive rats (SHR) were examined. Before i.c.v. d,l-propranolol or timolol, air stress increased renal activity (68% from 10.6 +/- 2.1 and 63% from 8.2 +/- 0.9 integrator resets/min respectively). In contrast, after i.c.v. d,l-propranolol or timolol in the same conscious SHR, air stress had no effect on renal sympathetic nerve activity (+7% from 8.1 +/- 1.7 and +7% from 5.5 +/- 1.0 integrator resets/min respectively). Air stress decreased UNaV in conscious SHR given i.c.v. saline vehicle (25% from 2.8 +/- 0.5 microEq/min/100 g body weight), but had no effect on effective renal plasma flow or glomerular filtration rate. In contrast, after i.c.v. d,l-propranolol or timolol, air stress had no effect on UNaV (0% from 2.8 +/- 0.5 and +9% from 3.3 +/- 0.3 microEq/min/100 g body weight respectively). Mean arterial pressure increased similarly during air stress with i.c.v. saline-vehicle or beta-adrenergic receptor antagonists. Intravenous administration of the same doses of d,l-propranolol or timolol did not prevent the increased renal sympathetic nerve activity or decreased UNaV responses resulting from air stress. These results suggest that central nervous system beta-adrenergic receptors mediate the increased renal sympathetic nerve activity and decreased UNaV responses resulting from stressful environmental stimulation in conscious SHR.

  12. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

    PubMed Central

    1983-01-01

    At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis. PMID:6309857

  13. Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

    PubMed Central

    Talamillo, Ana; Herboso, Leire; Pirone, Lucia; Pérez, Coralia; González, Monika; Sánchez, Jonatan; Mayor, Ugo; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S.; Sutherland, James D.; Barrio, Rosa

    2013-01-01

    SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues. PMID:23637637

  14. Desensitization of histamine H1 receptor-mediated inositol phosphate production in HeLa cells.

    PubMed Central

    Bristow, D. R.; Zamani, M. R.

    1993-01-01

    1. Histamine stimulated the accumulation of total [3H]-inositol phosphates (IPn) in control HeLa cells with an EC50 of 3.7 +/- 0.7 microM in the presence of 10 mM LiCl. The maximum response to histamine after 15 min incubation was 43 +/- 5% over basal accumulation and occurred at a concentration of 1 mM histamine. 2. The histamine-induced IPn production in HeLa cells was confirmed as H1 receptor-mediated, since the H1 antagonist mepyramine (10(-6) M) inhibited the histamine response (10(-4) M) by 83 +/- 7%, whereas the H2 antagonist, ranitidine (10(-4) M), and H3 antagonist, thioperamide (10(-6) M), were ineffective. 3. Histamine (10(-4) M) pretreatment of HeLa cells for 30 min desensitized the subsequent histamine-induced IPn accumulation. The desensitized cells accumulated IPn in response to histamine with an EC50 of 1.7 +/- 0.7 microM after 15 min incubation. The maximum histamine-induced IPn accumulation at 10(-4) M was 19 +/- 5% over basal and was significantly lower (P < 0.03) than the maximum response in control cells. 4. The desensitization of histamine-induced IPn accumulation was time-dependent and, at a desensitizing histamine concentration of 10(-4) M, the half-maximal attenuation occurred after approximately 9 min and maximum desensitization was achieved by 15-20 min. The desensitization of the IPn accumulation was a reversible phenomenon and full recovery of the response occurred 150 min after the removal of the desensitizing histamine-containing medium. The half-time for the recovery of the histamine-induced response was estimated at 120 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8358540

  15. 5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats.

    PubMed Central

    MacLean, M. R.; Sweeney, G.; Baird, M.; McCulloch, K. M.; Houslay, M.; Morecroft, I.

    1996-01-01

    1. We investigated 5-hydroxytryptamine (5-HT)-receptor mediated vasoconstriction in the main, first branch and resistance pulmonary arteries removed from control and pulmonary hypertensive rats. Contractile responses to 5-HT, 5-carboxamidotryptamine (5-CT, non-selective 5-HT1 agonist), and sumatriptan (5-HT1D-like receptor agonist) were studied. The effects of methiothepin (non-selective 5-HT1 + 2-receptor antagonist) and ketanserin (5-HT2A receptor antagonist) and GR55562 (a novel selective 5-HT1D receptor antagonist) on 5-HT-mediated responses were also studied. Basal levels of adenosine 3':5'-cyclic monophosphate ([cyclic AMP]i) and guanosine 3':5'-cyclic monophosphate ([cyclic GMP]i) were determined and we assessed the degree of inherent tone in the vessels under study. 2. 5-HT was most potent in the resistance arteries. pEC50 values were 5.6 +/- 0.1, 5.3 +/- 0.1, 5.0 +/- 0.2 in the resistance arteries, pulmonary branch and main pulmonary artery, respectively (n = at least 5 from 5 animals). The sensitivity to, and maximum response of, 5-HT was increased in all the arteries removed from the chronic hypoxic (CH) rats. In CH rats the pEC50 values were 5.9 +/- 0.2, 6.3 +/- 0.2, 6.4 +/- 0.2 and the increase in the maximum response was 35%, 51% and 41% in the resistance arteries, pulmonary branch and main pulmonary artery, respectively. Sumatriptan did not contract any vessel from the control rats whilst 5-CT did contract the resistance arteries. In the CH rats, however, they both contracted the resistance arteries (responses to sumatriptan were small) (pEC50: 5-CT; 5.4 +/- 0.2) and the pulmonary artery branches (pEC50: sumatriptan, 5.4 +/- 0.2; 5-CT, 5.4 +/- 0.2). 5-CT also caused a contraction in the main pulmonary artery (pEC50: 6.0 +/- 0.3). 3. Ketanserin (1 nM-1 microM) caused a competitive antagonism of the 5-HT response in all vessels tested. In control rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary

  16. P2X and P2Y Receptors—Role in the Pathophysiology of the Nervous System

    PubMed Central

    Puchałowicz, Kamila; Tarnowski, Maciej; Baranowska-Bosiacka, Irena; Chlubek, Dariusz; Dziedziejko, Violetta

    2014-01-01

    Purinergic signalling plays a crucial role in proper functioning of the nervous system. Mechanisms depending on extracellular nucleotides and their P2 receptors also underlie a number of nervous system dysfunctions. This review aims to present the role of purinergic signalling, with particular focus devoted to role of P2 family receptors, in epilepsy, depression, neuropathic pain, nervous system neoplasms, such as glioma and neuroblastoma, neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. The above-mentioned conditions are associated with changes in expression of extracellular ectonucleotidases, P2X and P2Y receptors in neurons and glial cells, as well as releasing considerable amounts of nucleotides from activated or damaged nervous tissue cells into the extracellular space, which contributes to disturbance in purinergic signalling. The numerous studies indicate a potential possibility of using synthetic agonists/antagonists of P2 receptors in treatment of selected nervous system diseases. This is of particular significance, since numerous available agents reveal a low effectiveness and often produce side effects. PMID:25530618

  17. Optogenetic evocation of field inhibitory postsynaptic potentials in hippocampal slices: a simple and reliable approach for studying pharmacological effects on GABAA and GABAB receptor-mediated neurotransmission

    PubMed Central

    Dine, Julien; Kühne, Claudia; Deussing, Jan M.; Eder, Matthias

    2014-01-01

    The GABAergic system is the main source of inhibition in the mammalian brain. Consequently, much effort is still made to develop new modulators of GABAergic synaptic transmission. In contrast to glutamatergic postsynaptic potentials (PSPs), accurate monitoring of GABA receptor-mediated PSPs (GABAR-PSPs) and their pharmacological modulation in brain tissue invariably requires the use of intracellular recording techniques. However, these techniques are expensive, time- and labor-consuming, and, in case of the frequently employed whole-cell patch-clamp configuration, impact on intracellular ion concentrations, signaling cascades, and pH buffering systems. Here, we describe a novel approach to circumvent these drawbacks. In particular, we demonstrate in mouse hippocampal slices that selective optogenetic activation of interneurons leads to prominent field inhibitory GABAAR- and GABABR-PSPs in area CA1 which are easily and reliably detectable by a single extracellular recording electrode. The field PSPs exhibit typical temporal and pharmacological characteristics, display pronounced paired-pulse depression, and remain stable over many consecutive evocations. Additionally validating the methodological value of this approach, we further show that the neuroactive steroid 5α-THDOC (5 μM) shifts the inhibitory GABAAR-PSPs towards excitatory ones. PMID:24478627

  18. The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle

    PubMed Central

    Welc, Steven S.; Morse, Deborah A.; Mattingly, Alex J.; Laitano, Orlando; King, Michelle A.; Clanton, Thomas L.

    2016-01-01

    In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic

  19. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  20. Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

    PubMed

    Shcherbatko, Anatoly; Foletti, Davide; Poulsen, Kris; Strop, Pavel; Zhu, Guoyun; Hasa-Moreno, Adela; Melton Witt, Jody; Loo, Carole; Krimm, Stellanie; Pios, Ariel; Yu, Jessica; Brown, Colleen; Lee, John K; Stroud, Robert; Rajpal, Arvind; Shelton, David

    2016-06-01

    Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications. PMID:27129281

  1. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  2. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  3. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  4. Accelerated FoxP2 evolution in echolocating bats.

    PubMed

    Li, Gang; Wang, Jinhong; Rossiter, Stephen J; Jones, Gareth; Zhang, Shuyi

    2007-01-01

    FOXP2 is a transcription factor implicated in the development and neural control of orofacial coordination, particularly with respect to vocalisation. Observations that orthologues show almost no variation across vertebrates yet differ by two amino acids between humans and chimpanzees have led to speculation that recent evolutionary changes might relate to the emergence of language. Echolocating bats face especially challenging sensorimotor demands, using vocal signals for orientation and often for prey capture. To determine whether mutations in the FoxP2 gene could be associated with echolocation, we sequenced FoxP2 from echolocating and non-echolocating bats as well as a range of other mammal species. We found that contrary to previous reports, FoxP2 is not highly conserved across all nonhuman mammals but is extremely diverse in echolocating bats. We detected divergent selection (a change in selective pressure) at FoxP2 between bats with contrasting sonar systems, suggesting the intriguing possibility of a role for FoxP2 in the evolution and development of echolocation. We speculate that observed accelerated evolution of FoxP2 in bats supports a previously proposed function in sensorimotor coordination.

  5. FoxP2 in songbirds.

    PubMed

    Wohlgemuth, Sandra; Adam, Iris; Scharff, Constance

    2014-10-01

    Humans with mutations in the transcription factor FOXP2 display a severe speech disorder. Songbirds are a powerful model system to study FoxP2. Like humans, songbirds communicate via vocalizations that are imitatively learned during critical periods and this learning is influenced by social factors and relies on functionally lateralized neural circuits. During the past five years significant progress has been made moving from a descriptive to a more mechanistic understanding of how FoxP2 functions in songbirds. Current evidence from molecular and electrophysiological studies indicates that FoxP2 is important for shaping synaptic plasticity of specific neuron populations. One future goal will be to identify the transcriptional regulation orchestrated by FoxP2 and its associated molecular network that brings about these physiological effects. This will be key to further unravel how FoxP2 influences synaptic function and thereby contributes to auditory guided vocal motor behavior in the songbird model.

  6. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

    PubMed

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

  7. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  8. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

    PubMed Central

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2012-01-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. PMID:16603603

  9. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    PubMed

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials.

  10. Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

    PubMed Central

    Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

    2016-01-01

    Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

  11. The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

    PubMed Central

    Batty, I H; Downes, C P

    1994-01-01

    Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively

  12. Androgen receptor-mediated non-genomic regulation of prostate cancer cell proliferation

    PubMed Central

    Liao, Ross S.; Ma, Shihong; Miao, Lu; Li, Rui; Yin, Yi

    2013-01-01

    Androgen receptor (AR)-mediated signaling is necessary for prostate cancer cell proliferation and an important target for therapeutic drug development. Canonically, AR signals through a genomic or transcriptional pathway, involving the translocation of androgen-bound AR to the nucleus, its binding to cognate androgen response elements on promoter, with ensuing modulation of target gene expression, leading to cell proliferation. However, prostate cancer cells can show dose-dependent proliferation responses to androgen within minutes, without the need for genomic AR signaling. This proliferation response known as the non-genomic AR signaling is mediated by cytoplasmic AR, which facilitates the activation of kinase-signaling cascades, including the Ras-Raf-1, phosphatidyl-inositol 3-kinase (PI3K)/Akt and protein kinase C (PKC), which in turn converge on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation, leading to cell proliferation. Further, since activated ERK may also phosphorylate AR and its coactivators, the non-genomic AR signaling may enhance AR genomic activity. Non-genomic AR signaling may occur in an ERK-independent manner, via activation of mammalian target of rapamycin (mTOR) pathway, or modulation of intracellular Ca2+ concentration through plasma membrane G protein-coupled receptors (GPCRs). These data suggest that therapeutic strategies aimed at preventing AR nuclear translocation and genomic AR signaling alone may not completely abrogate AR signaling. Thus, elucidation of mechanisms that underlie non-genomic AR signaling may identify potential mechanisms of resistance to current anti-androgens and help developing novel therapies that abolish all AR signaling in prostate cancer. PMID:26816736

  13. Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway.

    PubMed

    Xu, Xiaohong; Lu, Yang; Zhang, Guangxia; Chen, Lei; Tian, Dong; Shen, Xiuying; Yang, Yanling; Dong, Fanni

    2014-02-01

    Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30min. The purpose of the present study was further to investigate the effects of BPA for 24h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5d in vitro, the hippocampal neurons from 24h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100nM) or 17β-estradiol (17β-E2, 10nM) for 24h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen-activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17β-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

  14. Human Milk Components Modulate Toll-Like Receptor-Mediated Inflammation.

    PubMed

    He, YingYing; Lawlor, Nathan T; Newburg, David S

    2016-01-01

    Toll-like receptor (TLR) signaling is central to innate immunity. Aberrant expression of TLRs is found in neonatal inflammatory diseases. Several bioactive components of human milk modulate TLR expression and signaling pathways, including soluble toll-like receptors (sTLRs), soluble cluster of differentiation (sCD) 14, glycoproteins, small peptides, and oligosaccharides. Some milk components, such as sialyl (α2,3) lactose and lacto-N-fucopentaose III, are reported to increase TLR signaling; under some circumstances this might contribute toward immunologic balance. Human milk on the whole is strongly anti-inflammatory, and contains abundant components that depress TLR signaling pathways: sTLR2 and sCD14 inhibit TLR2 signaling; sCD14, lactadherin, lactoferrin, and 2'-fucosyllactose attenuate TLR4 signaling; 3'-galactosyllactose inhibits TLR3 signaling, and β-defensin 2 inhibits TLR7 signaling. Feeding human milk to neonates decreases their risk of sepsis and necrotizing enterocolitis. Thus, the TLR regulatory components found in human milk hold promise as benign oral prophylactic and therapeutic treatments for the many gastrointestinal inflammatory disorders mediated by abnormal TLR signaling.

  15. Maltodextrin and fat preference deficits in "taste-blind" P2X2/P2X3 knockout mice.

    PubMed

    Sclafani, Anthony; Ackroff, Karen

    2014-07-01

    Adenosine triphosphate is a critical neurotransmitter in the gustatory response to the 5 primary tastes in mice. Genetic deletion of the purinergic P2X2/P2X3 receptor greatly reduces the neural and behavioral response to prototypical primary taste stimuli. In this study, we examined the behavioral response of P2X double knockout mice to maltodextrin and fat stimuli, which appear to activate additional taste channels. P2X double knockout and wild-type mice were given 24-h choice tests (vs. water) with ascending concentrations of Polycose and Intralipid. In Experiment 1, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.5-4%) Polycose solutions but preferred concentrated (8-32%) Polycose to water. In a retest, the Polycose-experienced double knockout mice, like wild-type mice, preferred all Polycose concentrations. In Experiment 2, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.313-2.5%) Intralipid emulsions but preferred concentrated (5-20%) Intralipid to water. In a retest, the fat-experienced double knockout mice, like wild-type mice, strongly preferred 0.313-5% Intralipid to water. These results indicate that the inherent preferences of mice for maltodextrin and fat are dependent upon adenosine triphosphate taste cell signaling. With experience, however, P2X double knockout mice develop strong preferences for the nontaste flavor qualities of maltodextrin and fat conditioned by the postoral actions of these nutrients.

  16. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  17. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    PubMed Central

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  18. A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis*S⃞

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Girard-Dias, Wendell; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Docampo, Roberto; Alonso, Guillermo D.

    2008-01-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking. PMID:18801733

  19. Inhibition of isoproterenol-induced lipolysis in rat inguinal adipocytes in vitro by physiological melatonin via a receptor-mediated mechanism.

    PubMed

    Zalatan, F; Krause, J A; Blask, D E

    2001-09-01

    Because the pineal hormone melatonin has been implicated in affecting adiposity in rats and fatty acid transport in certain rat tumor models, we tested whether melatonin regulates lipolysis in a normal cell system in vitro. Adipocytes were isolated from the inguinal fat pads (i.e. sc fat) of Sprague Dawley male rats during mid-light phase. Lipolysis was stimulated with isoproterenol (3 microM), and cells were incubated for 4 h in the presence or absence of a physiological circulating concentration of melatonin (1 nM). Lipolysis was measured by determining the amount of glycerol present in the incubation buffer, expressed as nmol glycerol/mg cellular fatty acid. We observed a 20- to 30-fold stimulation of basal lipolysis by isoproterenol, and this stimulation was inhibited 50--70% by melatonin. Melatonin exhibited this effect over a wide range of concentrations tested (100 pM-1 microM) with an IC(50) of approximately 500 pM. The effect by melatonin (1 nM) was completely blocked by pertussis toxin (50 ng/ml), by 8-bromo-cAMP (10 nM), and by the melatonin receptor antagonist S-20928 (1 nM). These results suggest that the antilipolytic effect occurs through one of the G(i) protein-coupled melatonin receptors because we have shown that both the mt(1) (Mel 1a) and MT(2) (Mel 1b) melatonin receptors are expressed in inguinal adipocytes. Melatonin inhibition of lipolysis was not observed in adipocytes isolated from rat epididymal fat pads (i.e. visceral fat), even though these cells also express both the mt(1) and MT(2) receptors. The results indicate that physiological circulating concentrations of melatonin inhibit isoproterenol-induced lipolysis in rat adipocytes via a G protein-coupled melatonin receptor-mediated signal transduction pathway in a site-specific manner.

  20. Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation.

    PubMed

    Yin, Xiao-Hui; Zhang, Quan-Guang; Miao, Bei; Zhang, Guang-Yi

    2005-05-01

    A number of works show that the mitogen-activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed-lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.

  1. Increased desensitization of dopamine D₂ receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

    PubMed

    Al-Hasani, R; Foster, J D; Metaxas, A; Ledent, C; Hourani, S M O; Kitchen, I; Chen, Y

    2011-09-01

    G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D₂ receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D₂ receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D₂ receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D₂ receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D₂ receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D₂ receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D₂ receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine

  2. The oxidized low-density lipoprotein receptor mediates vascular effects of inhaled vehicle emissions

    EPA Science Inventory

    Rationale: To determine vascular signaling pathways involved in air pollution (vehicular engine emission) exposure -induced exacerbation of atherosclerosis, associated with onset of clinical cardiovascular events. Objective: To elucidate the role of oxidized LDL (oxLDL) and its ...

  3. Multi-functionalized hyaluronic acid nanogels crosslinked with carbon dots as dual receptor-mediated targeting tumor theranostics.

    PubMed

    Jia, Xu; Han, Yu; Pei, Mingliang; Zhao, Xubo; Tian, Kun; Zhou, Tingting; Liu, Peng

    2016-11-01

    Hyaluronic acid (HA)-based theranostic nanogels were designed for the tumor diagnosis and chemotherapy, by crosslinking the folate-terminated poly(ethylene glycol) modified hyaluronic acid (FA-PEG-HA) with carbon dots (CDs) for the first time. Due to the extraordinary fluorescence property of the integrated CDs, the theranostic nanogels could be used for the real-time and noninvasive location tracking to cancer cells. HA could load Doxorubicin (DOX) via electrostatic interaction with a drug-loading capacity (DLC) of 32.5%. The nanogels possessed an ideal release of DOX in the weak acid environment, while it was restrained in the neutral media, demonstrating the pH-responsive controlled release behavior. The cytotoxicity and cellular uptake results clearly illustrated that most DOX was released and accumulated in the cell nuclei and killed the cancer cells efficaciously, due to their dual receptor-mediated targeting characteristics. PMID:27516286

  4. The influence of receptor-mediated interactions on reaction-diffusion mechanisms of cellular self-organisation.

    PubMed

    Klika, Václav; Baker, Ruth E; Headon, Denis; Gaffney, Eamonn A

    2012-04-01

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction. PMID:22072186

  5. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    SciTech Connect

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.

    1987-09-07

    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  6. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    PubMed Central

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  7. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  8. The Molecular Mechanism of P2Y1 Receptor Activation

    PubMed Central

    Chan, H. C. Stephen; Vogel, Horst; Filipek, Slawomir

    2016-01-01

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1R) is activated by adenosine 5’-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1R activation. PMID:27460867

  9. P2X4R+ microglia drive neuropathic pain

    PubMed Central

    Beggs, Simon; Trang, Tuan; Salter, Michael W

    2016-01-01

    Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R+ microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition. PMID:22837036

  10. Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

    PubMed

    Haxholm, Gitte W; Nikolajsen, Louise F; Olsen, Johan G; Fredsted, Jacob; Larsen, Flemming H; Goffin, Vincent; Pedersen, Stine F; Brooks, Andrew J; Waters, Michael J; Kragelund, Birthe B

    2015-06-15

    Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

  11. Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells.

    PubMed

    Clark, Sara; Rainville, Jennifer; Zhao, Xing; Katzenellenbogen, Benita S; Pfaff, Donald; Vasudevan, Nandini

    2014-01-01

    While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17β-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Gαq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERα phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERα is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

  12. Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor.

    PubMed

    Lecat, Sandra; Belemnaba, Lazare; Galzi, Jean-Luc; Bucher, Bernard

    2015-07-01

    Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.

  13. Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents.

    PubMed

    Horváth, Gergely; Gölöncsér, Flóra; Csölle, Cecilia; Király, Kornél; Andó, Rómeó D; Baranyi, Mária; Koványi, Bence; Máté, Zoltán; Hoffmann, Kristina; Algaier, Irina; Baqi, Younis; Müller, Christa E; Von Kügelgen, Ivar; Sperlágh, Beáta

    2014-10-01

    In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways.

  14. Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents

    PubMed Central

    Horváth, Gergely; Gölöncsér, Flóra; Csölle, Cecilia; Király, Kornél; Andó, Rómeó D.; Baranyi, Mária; Koványi, Bence; Máté, Zoltán; Hoffmann, Kristina; Algaier, Irina; Baqi, Younis; Müller, Christa E.; Von Kügelgen, Ivar; Sperlágh, Beáta

    2014-01-01

    In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3 mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways. PMID:24971933

  15. Data Sharing in P2P Systems

    NASA Astrophysics Data System (ADS)

    Hayek, Rabab; Raschia, Guillaume; Valduriez, Patrick; Mouaddib, Noureddine

    In this chapter, we survey P2P data sharing systems. All along, we focus on the evolution from simple file-sharing systems, with limited functionalities, to Peer Data Management Systems (PDMS) that support advanced applications with more sophisticated data management techniques. Advanced P2P applications are dealing with semantically rich data (e.g., XML documents, relational tables), using a high-level SQL-like query language. We start our survey with an overview over the existing P2P network architectures, and the associated routing protocols. Then, we discuss data indexing techniques based on their distribution degree and the semantics they can capture from the underlying data. We also discuss schema management techniques which allow integrating heterogeneous data. We conclude by discussing the techniques proposed for processing complex queries (e.g., range and join queries). Complex query facilities are necessary for advanced applications which require a high level of search expressiveness. This last part shows the lack of querying techniques that allow for an approximate query answering.

  16. Kainate Receptors Mediate Synaptic Input to Transient and Sustained OFF Visual Pathways in Primate Retina

    PubMed Central

    Percival, Kumiko A.; Venkataramani, Sowmya; Gayet-Primo, Jacqueline; Grünert, Ulrike; Taylor, W. Rowland

    2014-01-01

    Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels. PMID:24872565

  17. Histamine H2 Receptor-Mediated Suppression of Intestinal Inflammation by Probiotic Lactobacillus reuteri

    PubMed Central

    Gao, Chunxu; Major, Angela; Rendon, David; Lugo, Monica; Jackson, Vanessa; Shi, Zhongcheng; Mori-Akiyama, Yuko

    2015-01-01

    ABSTRACT Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 [IL-6] and IL-1β in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [18F]fluorodeoxyglucose ([18F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc+ L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon. PMID:26670383

  18. The wedelolactone derivative inhibits estrogen receptor-mediated breast, endometrial, and ovarian cancer cells growth.

    PubMed

    Xu, Defeng; Lin, Tzu-Hua; Yeh, Chiuan-Ren; Cheng, Max A; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.

  19. Molecular Pathways: AXL, a Membrane Receptor Mediator of Resistance to Therapy

    PubMed Central

    Scaltriti, Maurizio; Elkabets, Moshe; Baselga, José

    2016-01-01

    AXL is a tyrosine kinase membrane receptor that signals via PI3K, MAPK, and protein kinase C (PKC), among other pathways. AXL has oncogenic potential and interacts with other membrane receptors, depending on their relative abundance and availability. The increased expression of AXL in cancer is often the result of pharmacologic selective pressure to a number of chemotherapies and targeted therapies and acts as a mechanism of acquired drug resistance. This resistance phenotype, frequently accompanied by epithelial-to-mesenchymal transition, can be reversed by AXL inhibition. In tumors with high levels of EGFR, including lung, head and neck, and triple-negative breast cancer, AXL dimerizes with this receptor and initiates signaling that circumvents the antitumor effects of anti-EGFR therapies. Likewise, AXL overexpression and dimerization with EGFR can overcome PI3K inhibition by activating the phospholipase C-γ-PKC cascade that, in turn, sustains mTORC1 activity. The causative role of AXL in inducing drug resistance is underscored by the fact that the suppression of AXL restores sensitivity to these agents. Hence, these observations indicate that AXL is selectively expressed in tumor cells refractory to therapy and that cotargeting AXL in this setting would potentially overcome drug resistance. The use of AXL inhibitors should be considered in the clinic. PMID:26763248

  20. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    NASA Astrophysics Data System (ADS)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  1. Characterization of the 5-hydroxytryptamine receptors mediating contraction in the pig isolated intravesical ureter

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Simonsen, Ulf; Recio, Paz; Rivera, Luis; Martínez, Ana Cristina; García-Sacristán, Albino; Orensanz, Luis M; Prieto, Dolores

    2003-01-01

    This study was designed to investigate the effect of 5-hydroxytryptamine (5-HT) and to characterize the 5-HT receptors involved in 5-HT responses in the pig intravesical ureter. 5-HT (0.01–10 μM) concentration-dependently increased the tone of intravesical ureteral strips, whereas the increases in phasic contractions were concentration-independent. The 5-HT2 receptor agonist α-methyl 5-HT, mimicked the effect on tone whereas weak or no response was obtained with 5-CT, 8-OH-DPAT, m-chlorophenylbiguanide and RS 67333, 5-HT1, 5-HT1A, 5-HT3 and 5-HT4 receptor agonists, respectively. 5-HT did not induce relaxation of U46619-contracted ureteral preparations. Pargyline (100 μM), a monoaminooxidase A/B activity inhibitor, produced leftward displacements of the concentration-response curves for 5-HT. 5-HT-induced tone was reduced by the 5-HT2 and 5-HT2A receptor antagonists ritanserine (0.1 μM) and spiperone (0.2 μM), respectively. However, 5-HT contraction was not antagonized by cyanopindolol (2 μM), SDZ–SER 082 (1 μM), Y-25130 (1 μM) and GR 113808 (0.1 μM), which are respectively, 5-HT1A/1B, 5-HT2B/2C, 5-HT3, and 5-HT4 selective receptor antagonists. Removal of the urothelium did not modify 5-HT-induced contractions. Blockade of neuronal voltage-activated sodium channels, α-adrenergic receptors and adrenergic neurotransmission with tetrodotoxin (1 μM), phentolamine (0.3 μM) and guanethidine (10 μM), respectively, reduced the contractions to 5-HT. However, physostigmine (1 μM), atropine (0.1 μM) and suramin (30 μM), inhibitors of cholinesterase activity, muscarinic- and purinergic P2-receptors, respectively, failed to modify the contractions to 5-HT. These results suggest that 5-HT increases the tone of the pig intravesical ureter through 5-HT2A receptors located at the smooth muscle. Part of the 5-HT contraction is indirectly mediated via noradrenaline release from sympathetic nerves. PMID:12522083

  2. Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression.

    PubMed

    de la Garza-Rodea, Anabel S; Baldwin, Dianna M; Oskouian, Babak; Place, Robert F; Bandhuvula, Padmavathi; Kumar, Ashok; Saba, Julie D

    2014-01-01

    S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle differentiation, homeostasis, and satellite cell (SC) activation. By regulating S1P levels, SPL also controls SC recruitment and muscle regeneration, representing a potential therapeutic target for muscular dystrophy. We found that SPL is induced during myoblast differentiation. To investigate SPL's role in myogenesis at the cellular level, we generated and characterized a murine myoblast SPL-knockdown (SPL-KD) cell line lacking SPL. SPL-KD cells accumulated intracellular and extracellular S1P and failed to form myotubes under conditions that normally stimulate myogenic differentiation. Under differentiation conditions, SPL-KD cells also demonstrated delayed induction of 3 myogenic microRNAs (miRNAs), miR-1, miR-206, and miR-486. SPL-KD cells successfully differentiated when treated with an S1P1 agonist, S1P2 antagonist, and combination treatments, which also increased myogenic miRNA levels. SPL-KD cells transfected with mimics for miR-1 or miR-206 also overcame the differentiation block. Thus, we show for the first time that the S1P/SPL/S1P-receptor axis regulates the expression of a number of miRNAs, thereby contributing to myogenic differentiation.

  3. Dopamine D2 Receptor-Mediated Regulation of Pancreatic β Cell Mass.

    PubMed

    Sakano, Daisuke; Choi, Sungik; Kataoka, Masateru; Shiraki, Nobuaki; Uesugi, Motonari; Kume, Kazuhiko; Kume, Shoen

    2016-07-12

    Understanding the molecular mechanisms that regulate β cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances β cell mass. Over time, islet β cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of β cells through increasing intracellular cAMP. DPD prevented β cell dedifferentiation, which together highly contributed to the increased β cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of β cells. Drd2 overexpression sensitized the dopamine responsiveness of β cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5'-N-ethylcarboxamidoadenosine, a previously identified promoter of β cell proliferation, acted with DPD to increase the number of β cells. In humans, dopamine also modulates β cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling. PMID:27373926

  4. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells

    PubMed Central

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-won

    2016-01-01

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7–8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  5. The prolactin receptor mediates HOXA1-stimulated oncogenicity in mammary carcinoma cells.

    PubMed

    Hou, Lin; Xu, Bing; Mohankumar, Kumarasamypet M; Goffin, Vincent; Perry, Jo K; Lobie, Peter E; Liu, Dong-Xu

    2012-12-01

    The HOX genes are a highly conserved subgroup of homeodomain-containing transcription factors that are crucial to normal development. Forced expression of HOXA1 results in oncogenic transformation of immortalized human mammary cells with aggressive tumour formation in vivo. Microarray analysis identified that the prolactin receptor (PRLR) was significantly upregulated by forced expression of HOXA1 in mammary carcinoma cells. To determine prolactin (PRL) involvement in HOXA1‑induced oncogenicity in mammary carcinoma cells (MCF-7), we examined the effect of human prolactin (hPRL)-initiated PRLR signal transduction on changes in cellular behaviour mediated by HOXA1. Forced expression of HOXA1 in MCF-7 cells increased PRLR mRNA and protein expression. Forced expression of HOXA1 also enhanced hPRL-stimulated phosphorylation of both STAT5A/B and p44/42 MAPK, and increased subsequent transcriptional activity of STAT5A and STAT5B, and Elk-1 and Sap1a, respectively. Moreover, forced expression of HOXA1 in MCF-7 cells enhanced the hPRL‑stimulated increase in total cell number as a consequence of enhanced cell proliferation and cell survival, and also enhanced hPRL-stimulated anchorage-independent growth in soft agar. Increased anchorage-independent growth was attenuated by the PRLR antagonist ∆1-9-G129R‑hPRL. In conclusion, we have demonstrated that HOXA1 increases expression of the cell surface receptor PRLR and enhances PRLR-mediated signal transduction. Thus, the PRLR is one mediator of HOXA1‑stimulated oncogenicity in mammary carcinoma cells. PMID:23064471

  6. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells.

    PubMed

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-Won

    2016-08-31

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  7. Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

    PubMed

    Park, Se-Ra; Kim, Dong-Jae; Han, Seung-Hyun; Kang, Min-Jung; Lee, Jun-Young; Jeong, Yu-Jin; Lee, Sang-Jin; Kim, Tae-Hyoun; Ahn, Sang-Gun; Yoon, Jung-Hoon; Park, Jong-Hwan

    2014-05-01

    Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

  8. Ionotropic glutamate receptors mediate inducible defense in the water flea Daphnia pulex.

    PubMed

    Miyakawa, Hitoshi; Sato, Masanao; Colbourne, John K; Iguchi, Taisen

    2015-01-01

    Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, "neckteeth," in response to chemical cues or signals, referred to as "kairomones," in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the recognition

  9. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia.

    PubMed

    Qu, Wen-Sheng; Liu, Jun-Li; Li, Chun-Yu; Li, Xiao; Xie, Min-Jie; Wang, Wei; Tian, Dai-Shi

    2015-11-01

    Previous reports have suggested that epidermal growth factor receptor (EGFR) is involved in microglia activation characterized by cell morphology changes, cytokine production and cell migration; and the biochemical regulation of the microglia migration is a potential therapeutic target following CNS inflammatory damages. However, the role of EGFR in microglia motility after inflammatory stimulation remains unknown. In the present study, lipopolysaccharide (LPS) was found to trigger rapid EGFR phosphorylation within 10 min, which was sustained during long-term stimulation in both primary microglial cells and the cultured BV2 microglial cells, furthermore, blocking EGFR phosphorylation by AG1478 significantly attenuated the LPS-induced chemotactic and chemokinetic migration of microglia. In addition, LPS could initiate calcium oscillation in microglia during live-cell recording, however, an intracellular calcium chelator and a selective inhibitor of calcium/calmodulin-dependent protein kinase II, but not an extracellular calcium chelator, remarkably suppressed the LPS-induced EGFR phosphorylation in BV2 microglia cells. As EGFR is not a traditional receptor for LPS, these findings suggest that the rapid phosphorylation of EGFR is attributed to the LPS-triggered intracellular calcium mobilization. By examining the downstream signals of EGFR, we further proved that extracellular signal-regulated kinase (ERK) is essential for EGFR-mediated microglia migration, because ERK inhibition attenuated the chemotactic and chemokinetic migration of microglia that had been induced by either LPS or EGF. Collectively, these results suggest that LPS could trigger the rapid phosphorylation of EGFR and subsequent ERK activation through mobilizing calcium activity, which underlies the microglia migration in an inflammatory condition.

  10. The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis.

    PubMed

    Aguado, Tania; Romero, Eva; Monory, Krisztina; Palazuelos, Javier; Sendtner, Michael; Marsicano, Giovanni; Lutz, Beat; Guzmán, Manuel; Galve-Roperh, Ismael

    2007-08-17

    Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Excitotoxicity and stroke can induce neural progenitor (NP) proliferation and differentiation as an attempt of neuroregeneration after damage. Here we investigated the mechanism of hippocampal progenitor cell engagement upon excitotoxicity induced by kainic acid administration and the putative involvement of the CB1 cannabinoid receptor in this process. Adult NPs express kainate receptors that mediate proliferation and neurosphere generation in vitro via CB1 cannabinoid receptors. Similarly, in vivo studies showed that excitotoxicity-induced hippocampal NPs proliferation and neurogenesis are abrogated in CB1-deficient mice and in wild-type mice administered with the selective CB1 antagonist rimonabant (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide; SR141716). Kainate stimulation increased basic fibroblast growth factor (bFGF) expression in cultured NPs in a CB1-dependent manner as this response was prevented by rimonabant and mimicked by endocannabinoids. Likewise, in vivo analyses showed that increased hippocampal expression of bFGF, as well as of brain-derived neurotrophic factor and epidermal growth factor, occurs upon excitotoxicity and that CB1 receptor ablation prevents this induction. Moreover, excitotoxicity increased the number of CB1+ bFGF+ cells, and this up-regulation preceded NP proliferation. In summary, our results show the involvement of the CB1 cannabinoid receptor in NP proliferation and neurogenesis induced by excitotoxic injury and support a role for bFGF signaling in this process.

  11. Ionotropic Glutamate Receptors Mediate Inducible Defense in the Water Flea Daphnia pulex

    PubMed Central

    Miyakawa, Hitoshi; Sato, Masanao; Colbourne, John K.; Iguchi, Taisen

    2015-01-01

    Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, “neckteeth,” in response to chemical cues or signals, referred to as “kairomones,” in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the

  12. Eclogitic pyroxenes, ordered with P2 symmetry

    USGS Publications Warehouse

    Clark, J.R.; Papike, J.J.

    1966-01-01

    X-ray diffraction crystal-structure analysis of omphacite from eclogite, Tiburon Peninsula, Marin County, California, shows that this clinopyroxene has P2 symmetry with a nearly ordered distribution of the multiple cation content defined by its approximate formula: (Na0.5Ca0.5) (Mg 0.4Fe2+0.1Al0.4Fe3+0.1)Si2O6. Na+ and Ca2+ tend to assume alternate locations in the structure, and (Mg,Fe2+) octahedra alternate with Al3+ or (Al,F3+) octahedra in chains along c.

  13. Electronic states of BP, BP +, BP -, B 2P 2, B2P2- and B2P2+

    NASA Astrophysics Data System (ADS)

    Linguerri, Roberto; Komiha, Najia; Oswald, Rainer; Mitrushchenkov, Alexander; Rosmus, Pavel

    2008-05-01

    Using augmented sextuple zeta basis sets and internally contracted multireference configuration interaction (MRCI) wavefunctions, potential energy, electric dipole and transition moments have been computed for the X 3Π, a 1Σ +, b 1Π and A 3Σ - states of BP, X 2Σ + and A 2Π states of BP - and X 4Σ - and A 4Π states of BP +. From these data spectroscopic constants, radiative transition probabilities and photoelectron spectra of BP - and BP have been evaluated. The non-vanishing spin-orbit coupling elements between the four low lying triplet and singlet states of the neutral BP have also been calculated from MRCI wavefunctions. The treatment of the corresponding perturbations in the manifold of dense rovibrational states in the three lowest states would require a precise knowledge of the electronic excitation energies. Our best singlet-triplet separations (X-a) are calculated to be 2412 cm -1 (MRCI) and 2482 cm -1 (restricted coupled cluster with perturbative triples (RCCSD(T))) with an estimated error bound of about ±200 cm -1. All three states have long radiative lifetimes with cascading among the rovibrational levels of different states. The ionization energy IE e of BP is calculated to be 9.22 eV (MRCI) and 9.48 eV (RCCSD(T)), the electron affinity EA e 2.51 eV (MRCI) and 2.74 eV (RCCSD(T)). The photoelectron spectra of BP and BP - have been obtained from the Franck-Condon factors of the MRCI potentials. For the UV spectroscopy the dipole allowed radiative transition probabilities are given for A 3Σ - ↔ X 3Π, b 1Π ↔ a 1Σ + of BP, A 2Π ↔ X 2Σ + of BP - and A 4Π ↔ X 4Σ - of BP +. The ionization energy IE e of B 2P 2 of 8.71 eV and the electron affinity EA e of 2.34 eV have been calculated by the RCCSD(T)/aVQZ approach. Also the harmonic vibrational wavenumbers for the electronic ground states of the ions B2P2+ and B2P2- are given.

  14. Regulation of P2X2 Receptors by the Neuronal Calcium Sensor VILIP1

    PubMed Central

    Chaumont, Severine; Compan, Vincent; Toulme, Estelle; Richler, Esther; Housley, Gary D.; Rassendren, Francois; Khakh, Baljit S.

    2012-01-01

    Extracellular adenosine triphosphate (ATP) activates P2X receptors, which are involved in diverse physiological functions. Using a proteomic approach, we identified the neuronal calcium sensor VILIP1 as interacting with P2X2 receptors. We found that VILIP1 forms a signaling complex in vitro and in vivo with P2X2 receptors and regulates P2X2 receptor sensitivity to ATP, peak response, surface expression, and diffusion. VILIP1 constitutively binds to P2X2 receptors and displays enhanced interactions in an activation- and calcium-dependent manner owing to exposure of its binding segment in P2X2 receptors. VILIP1-P2X2 interactions are also enhanced in hippocampal neurons during conditions of action potential firing known to trigger P2X2 receptor activation. Our data thus reveal a previously unrecognized function for the neuronal calcium sensor protein VILIP1 and a mechanism for regulation of ATP-dependent P2X receptor signaling by neuronal calcium sensors. PMID:18922787

  15. Group I metabotropic glutamate receptor mediated dynamic immune dysfunction in children with fragile X syndrome

    PubMed Central

    2014-01-01

    Background Fragile X syndrome (FXS) is the leading cause of inheritable intellectual disability in male children, and is predominantly caused by a single gene mutation resulting in expanded trinucleotide CGG-repeats within the 5’ untranslated region of the fragile X mental retardation (FMR1) gene. Reports have suggested the presence of immune dysregulation in FXS with evidence of altered plasma cytokine levels; however, no studies have directly assessed functional cellular immune responses in children with FXS. In order to ascertain if immune dysregulation is present in children with FXS, dynamic cellular responses to immune stimulation were examined. Methods Peripheral blood mononuclear cells (PBMC) were from male children with FXS (n = 27) and from male aged-matched typically developing (TD) controls (n = 8). PBMC were cultured for 48 hours in media alone or with lipopolysaccharides (LPS; 1 μg/mL) to stimulate the innate immune response or with phytohemagglutinin (PHA; 8 μg/mL) to stimulate the adaptive T-cell response. Additionally, the group I mGluR agonist, DHPG, was added to cultures to ascertain the role of mGluR signaling in the immune response in subject with FXS. Supernatants were harvested and cytokine levels were assessed using Luminex multiplexing technology. Results Children with FXS displayed similar innate immune response following challenge with LPS alone when compared with TD controls; however, when LPS was added in the presence of a group I mGluR agonist, DHPG, increased immune response were observed in children with FXS for a number of pro-inflammatory cytokines including IL-6 (P = 0.02), and IL-12p40 (P < 0.01). Following PHA stimulation, with or without DHPG, no significant differences between subjects with FXS and TD were seen. Conclusions In unstimulated cultures, subjects with FXS did not display altered dynamic immune response to LPS or PHA alone; however, subjects with FXS showed an altered response to co

  16. Platelet-Activating Factor Receptors Mediate Excitatory Postsynaptic Hippocampal Injury in Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Geathers, Jasmine S.; Allan, Kevin C.; Gelbard, Harris A.

    2016-01-01

    Gray matter degeneration contributes to progressive disability in multiple sclerosis (MS) and can occur out of proportion to measures of white matter disease. Although white matter pathology, including demyelination and axon injury, can lead to secondary gray matter changes, we hypothesized that neurons can undergo direct excitatory injury within the gray matter independent of these. We tested this using a model of experimental autoimmune encephalomyelitis (EAE) with hippocampal degeneration in C57BL/6 mice, in which immunofluorescent staining showed a 28% loss of PSD95-positive excitatory postsynaptic puncta in hippocampal area CA1 compared with sham-immunized controls, despite preservation of myelin and VGLUT1-positive excitatory axon terminals. Loss of postsynaptic structures was accompanied by appearance of PSD95-positive debris that colocalized with the processes of activated microglia at 25 d after immunization, and clearance of debris was followed by persistently reduced synaptic density at 55 d. In vitro, addition of activated BV2 microglial cells to hippocampal cultures increased neuronal vulnerability to excitotoxic dendritic damage following a burst of synaptic activity in a manner dependent on platelet-activating factor receptor (PAFR) signaling. In vivo treatment with PAFR antagonist BN52021 prevented PSD95-positive synapse loss in hippocampi of mice with EAE but did not affect development of EAE or local microglial activation. These results demonstrate that postsynaptic structures can be a primary target of injury within the gray matter in autoimmune neuroinflammatory disease, and suggest that this may occur via PAFR-mediated modulation of activity-dependent synaptic physiology downstream of microglial activation. SIGNIFICANCE STATEMENT Unraveling gray matter degeneration is critical for developing treatments for progressive disability and cognitive impairment in multiple sclerosis (MS). In a mouse model of MS, we show that neurons can undergo injury

  17. The positive feedback action of vasopressin on its own release from rat septal tissue in vitro is receptor-mediated.

    PubMed

    Landgraf, R; Ramirez, A D; Ramirez, V D

    1991-04-01

    The effect of arginine vasopressin (AVP) on its own septal release was evaluated using an in vitro superfusion procedure. As compared to basal release from septal fragments, pulses of synthetic AVP (15 pg/5 min) resulted in a 25-fold augmented release of endogenous AVP, indicating a positive feedback action. Both the basal and stimulated AVP release were significantly increased by 60 mM potassium and markedly reduced by omission of calcium. Preincubation of the septal fragments with the V2/V1 AVP receptor antagonist d(CH2)5 [D-Tyr (Et)2,Val4]AVP resulted in a dose-dependent inhibition of the positive feedback action of AVP which was nearly completely blocked at doses between 1.25 and 5 ng per 100 microliters incubation medium. As compared to this effect, the V1 antagonist d(CH2)5 Tyr (Me)2 AVP as well as oxytocin were significantly less potent. The results suggest that the positive feedback action of AVP on its own release from septal fragments is potassium-stimulated, calcium-dependent and mainly V2 receptor-mediated. The physiological significance of this phenomenon remains to be shown. PMID:1830507

  18. An special epithelial staining agents: folic acid receptor-mediated diagnosis (FRD) effectively and conveniently screen patients with cervical cancer.

    PubMed

    Lu, Meng-Han; Hu, Ling-Yun; Du, Xin-Xin; Yang, Min; Zhang, Wei-Yi; Huang, Ke; Li, Li-An; Jiang, Shu-Fang; Li, Ya-Li

    2015-01-01

    High-quality screening with cytology has markedly reduced mortality from cervical cancer. However, it needs experienced pathologists to review and make the final decisions. We have developed folic acid receptor-mediated diagnosis (FRD) kits to effectively and conveniently screen patients with cervical cancer. We conduct present study aim to assess clinical significances of FRD in screening cervical cancer. A total of 169 patients were enrolled at Chinese People's liberation Army (PLA) general hospital. We compared diagnostic significances of FRD with thinprep cytology test (TCT). Meanwhile, colposcopy was also performed to confirm any lesion suspicious for cervical cancer. The sensitivity and specificity of FRD were 71.93% and 66.07% in diagnosis cervical cancer, respectively. Meanwhile, the positive predictive values (PPV), negative predictive values (NPV), Youden index were 51.90%, 82.22%, 0.38, respectively. On the other hand, the sensitivity and specificity of TCT in diagnosis cervical cancer were 73.68% and 61.61% respectively. PPV, NPV and Youden index for TCT were 49.41%, 82.14% and 0.35 respectively. Overall, FRD have high values of sensitivity, specificity and Youden index. However, this difference failed to statistical significance. FRD have comparable diagnostic significance with TCT. Therefore, FRD might serve as one effective method to screen cervical cancer. Especially for those patients living in remote regions of China, where cytology was unavailable.

  19. Modulation of AMPA receptor mediated current by nicotinic acetylcholine receptor in layer I neurons of rat prefrontal cortex

    PubMed Central

    Tang, Bo; Luo, Dong; Yang, Jie; Xu, Xiao-Yan; Zhu, Bing-Lin; Wang, Xue-Feng; Yan, Zhen; Chen, Guo-Jun

    2015-01-01

    Layer I neurons in the prefrontal cortex (PFC) exhibit extensive synaptic connections with deep layer neurons, implying their important role in the neural circuit. Study demonstrates that activation of nicotinic acetylcholine receptors (nAChRs) increases excitatory neurotransmission in this layer. Here we found that nicotine selectively increased the amplitude of AMPA receptor (AMPAR)-mediated current and AMPA/NMDA ratio, while without effect on NMDA receptor-mediated current. The augmentation of AMPAR current by nicotine was inhibited by a selective α7-nAChR antagonist methyllycaconitine (MLA) and intracellular calcium chelator BAPTA. In addition, nicotinic effect on mEPSC or paired-pulse ratio was also prevented by MLA. Moreover, an enhanced inward rectification of AMPAR current by nicotine suggested a functional role of calcium permeable and GluA1 containing AMPAR. Consistently, nicotine enhancement of AMPAR current was inhibited by a selective calcium-permeable AMPAR inhibitor IEM-1460. Finally, the intracellular inclusion of synthetic peptide designed to block GluA1 subunit of AMPAR at CAMKII, PKC or PKA phosphorylation site, as well as corresponding kinase inhibitor, blocked nicotinic augmentation of AMPA/NMDA ratio. These results have revealed that nicotine increases AMPAR current by modulating the phosphorylation state of GluA1 which is dependent on α7-nAChR and intracellular calcium. PMID:26370265

  20. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations

    PubMed Central

    2016-01-01

    Lipid–polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle–membrane engagement, followed by a slow growth in the number of ligand–receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand–receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle–membrane interactions to occur even when the ligand density is low. The LPH–membrane avidity is enhanced by the increased stability of each receptor–ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle–cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest. PMID:25438167

  1. Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radial-flow detachment assay.

    PubMed Central

    Cozens-Roberts, C; Quinn, J A; Lauffenburger, D A

    1990-01-01

    Quantitative information regarding the kinetics of receptor-mediated cell adhesion to a ligand-coated surface are crucial for understanding the role of certain key parameters in many physiological and biotechnology-related processes. Here, we use the probabilistic attachment and detachment models developed in the preceding paper to interpret transient data from well-defined experiments. These data are obtained with a simple model cell system that consists of receptor-coated latex beads (prototype cells) and a Radial-Flow Detachment Assay (RFDA) using a ligand-coated glass disc. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine transient behavior with particles that possess fairly uniform properties that can be varied systematically, and the RFDA is designed for direct observation of adhesion to the ligand-coated glass surface over a range of shear stresses. Our experiments focus on the effects of surface shear stress, receptor density, and ligand density. These data provide a crucial test of the probabilistic framework. We show that these data can be explained with the probabilistic analyses, whereas they cannot be readily interpreted on the basis of a deterministic analysis. In addition, we examine transient data on cell adhesion reported from other assays, demonstrating the consistency of these data with the predictions of the probabilistic models. Images FIGURE 2 PMID:2174272

  2. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  3. Trans-activation of EphA4 and FGF receptors mediated by direct interactions between their cytoplasmic domains

    PubMed Central

    Yokote, Hideyuki; Fujita, Koji; Jing, Xuefeng; Sawada, Takahiro; Liang, Sitai; Yao, Li; Yan, Xiaomei; Zhang, Yueqiang; Schlessinger, Joseph; Sakaguchi, Kazushige

    2005-01-01

    A yeast two-hybrid analysis has shown that the juxtamembrane region of FGF receptor 3 (FGFR3) interacts with the cytoplasmic domain of EphA4, which is a member of the largest family of receptor tyrosine kinases. Complex formation between the two receptors was shown to be mediated by direct interactions between the juxtamembrane domain of FGFR1, FGFR2, FGFR3, or FGFR4 and the N-terminal portion of the tyrosine kinase domain of EphA4. Activation of FGFR1 in transfected cells resulted in tyrosine phosphorylation of a kinase-negative EphA4 mutant and activation of EphA4 led to tyrosine phosphorylation of a kinase-negative FGFR1 mutant. Moreover, both receptors stimulate tyrosine phosphorylation of the docking protein FRS2α and induce mitogen-activated protein kinase stimulation with a time course and intensity that depends on the ligand that is applied. We also demonstrate that FGF-receptor-mediated mitogen-activated protein kinase stimulation is potentiated in cells costimulated with ephrin-A1. The direct interaction between EphA4 and FGFRs and the potentiation of FGF response that is induced by ephrin-A1 stimulation may modulate the biological responses that are mediated by these receptor families in cells or tissues in which the two receptors are coexpressed. PMID:16365308

  4. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  5. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations

    NASA Astrophysics Data System (ADS)

    Li, Zhenlong; Gorfe, Alemayehu A.

    2014-12-01

    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  6. Histaminergic H1 receptors mediate L-histidine-induced anxiety in elevated plus-maze test in mice.

    PubMed

    Kumar, Kuchibhotla Vijaya; Krishna, Devarakonda Rama; Palit, Gautam

    2007-05-01

    The central histaminergic system is reported to mediate behavioural, hormonal and physiological homeostasis of living organisms. Recent reports indicate its prominent role in various neurobehavioural disorders such as depression and psychosis. This study evaluated the effect of activation of the central histaminergic system in anxiety-like conditions, using the elevated plus-maze test in mice, and elucidated the role of different histaminergic receptors mediating such effects. Peripheral administration of L-histidine (L-His), in a dose-dependent manner, significantly decreased the exploration time in open arms and number of entries into open arms without modifying the number of entries into closed arms of the elevated plus-maze, indicating anxiogenesis. Further, such effects of central histamine were significantly attenuated, in a dose-dependent manner, by pretreatment with pyrilamine (H1 receptor antagonist). Pretreatment with either zolantidine (H2 receptor antagonist) or thioperamide (H3 receptor antagonist), however, failed to attenuate the L-His-induced anxiogenesis. Our results indicate that anxiogenic effects of central histaminergic system appear to be mediated prominently by activation of H1 receptors.

  7. Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells

    SciTech Connect

    Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

    1988-02-01

    A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

  8. Fatty acyl specificity of the receptor-mediated release of polyunsaturated fatty acids from vascular endothelial cells

    SciTech Connect

    Rosenthal, M.D.

    1987-05-01

    Histamine and bradykinin appear to exhibit the same fatty acid specificity as thrombin. Incubation of human umbilical vein endothelial cells with 10 ..mu..M histamine for 10 min in buffered saline containing 50 ..mu..M fat-free albumin stimulates the release of previously incorporated (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) or (/sup 14/C)20:3(n-6). Similarly calf pulmonary artery endothelial cells release (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) in response to either bradykinin (1 /sup +/g/ml) or histamine (10..mu..M). In both types of endothelial cells, the calcium ionophore A23187 (10 ..mu..M) exhibits the same pattern of fatty acyl specificity as the receptor-mediated agonists. By contrast, mellitin (2-4 ..mu..g/ml) stimulates the release of free 22:4(n-6) and oleate in addition to arachidonate; release of 22:4(n-6) is 30-70% that of arachidonate. These results suggest that histamine, bradykinin and thrombin stimulate a common calcium-dependent fatty acyl-specific phospholipase activity.

  9. Resveratrol attenuates acute kidney injury by inhibiting death receptor-mediated apoptotic pathways in a cisplatin-induced rat model

    PubMed Central

    Hao, Qiufa; Xiao, Xiaoyan; Zhen, Junhui; Feng, Jinbo; Song, Chun; Jiang, Bei; Hu, Zhao

    2016-01-01

    Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti-inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin-induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: Control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor-α (TNF-α), caspase-8 and Bcl-2 associated protein X apoptosis regulator (Bax), and decreased expression of anti-apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl-2). Administration of resveratrol significantly reversed the cisplatin-induced alteration in these apoptosis-associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin-induced acute kidney injury through inactivation of the death receptor-mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury. PMID:27600998

  10. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  11. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations.

    PubMed

    Li, Zhenlong; Gorfe, Alemayehu A

    2015-01-14

    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  12. Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-Functionalized Radiolabeled Gold Nanoparticles in Tumor Mice.

    PubMed

    Silva, Francisco; Zambre, Ajit; Campello, Maria Paula Cabral; Gano, Lurdes; Santos, Isabel; Ferraria, Ana Maria; Ferreira, Maria João; Singh, Amolak; Upendran, Anandhi; Paulo, António; Kannan, Raghuraman

    2016-04-20

    To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of (67)Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7-14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice indicated that the presence of BBN influences to some degree the biological profile of the nanoconstructs. For IV administration, the receptor-mediated pathway appears to be outweighed by the EPR effect. By contrast, in IP administration, it is reasoned that the GRPr-mediated mechanism plays a role in pancreas uptake. PMID:27003101

  13. Receptor-mediated effects of nicotine and its nitrosated derivative NNK on pulmonary neuroendocrine cells.

    PubMed

    Schuller, Hildegard M; Plummer, Howard K; Jull, Brian A

    2003-01-01

    Pulmonary neuroendocrine cells (PNECs) have been implicated in the development of small cell lung carcinoma (SCLC) and pediatric asthma, and smoking is a risk factor for both diseases. We as well as others have shown that the alpha(7) nicotinic acetylcholine receptor (alpha(7) nAChR) regulates the release of 5-hydroxytryptamine (5-HT, serotonin) in PNECs and SCLC. Serotonin is an autocrine growth factor for PNECs and SCLC and acts as broncho-constrictor. We found that nicotine and its nitrosated carcinogenic derivative 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bind to the alpha(7) nAChR in SCLC and PNECs, resulting in the influx of Ca(2+), release of 5-HT, and activation of a mitogenic pathway mediated by protein kinase C (PKC), Raf-1, mitogen activated protein kinase (MAPK) and c-myc. Exposure to 10% CO(2) acted synergistically. Unstimulated SCLC cells from smokers demonstrated high base levels of 5-HT release and of individual downstream signaling components in comparison to PNECs. Subchronic exposure of PNECs to NNK up-regulated the alpha(7) nAChR and its associated serotonergic mitogenic pathway in PNECs, an effect that may contribute to the development of SCLC in smokers and pediatric asthma in children of mothers who smoke.

  14. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    PubMed

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M; Kaczmarek, Leonard K; Flavell, Richard A

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  15. Potential insight for drug discovery from high fidelity receptor-mediated transduction mechanisms in insects

    PubMed Central

    Raffa, Robert B.; Raffa, Kenneth F.

    2011-01-01

    Introduction There is a pervasive and growing concern about the small number of new pharmaceutical agents. There are many proposed explanations for this trend that do not involve the drug-discovery process per se, but the discovery process itself has also come under scrutiny. If the current paradigms are indeed not working, where are novel ideas to come from? Perhaps it is time to look to novel sources. Areas covered The receptor-signaling and 2nd-messenger transduction processes present in insects are quite similar to those in mammals (involving G proteins, ion channels, etc.). However, a review of these systems reveals an unprecedented degree of high potency and receptor selectivity to an extent greater than that modeled in most current drug-discovery approaches. Expert opinion A better understanding of insect receptor pharmacology could stimulate novel theoretical and practical ideas in mammalian pharmacology (drug discovery) and, conversely, the application of pharmacology and medicinal chemistry principles could stimulate novel advances in entomology (safer and more targeted control of pest species). PMID:21984882

  16. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

    PubMed

    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng

    2015-06-15

    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  17. Type Ib BMP receptors mediate the rate of commissural axon extension through inhibition of cofilin activity

    PubMed Central

    Yamauchi, Ken; Varadarajan, Supraja G.; Li, Joseph E.; Butler, Samantha J.

    2013-01-01

    Bone morphogenetic proteins (BMPs) have unexpectedly diverse activities establishing different aspects of dorsal neural circuitry in the developing spinal cord. Our recent studies have shown that, in addition to spatially orienting dorsal commissural (dI1) axons, BMPs supply ‘temporal’ information to commissural axons to specify their rate of growth. This information ensures that commissural axons reach subsequent signals at particular times during development. However, it remains unresolved how commissural neurons specifically decode this activity of BMPs to result in their extending axons at a specific speed through the dorsal spinal cord. We have addressed this question by examining whether either of the type I BMP receptors (Bmpr), BmprIa and BmprIb, have a role controlling the rate of commissural axon growth. BmprIa and BmprIb exhibit a common function specifying the identity of dorsal cell fate in the spinal cord, whereas BmprIb alone mediates the ability of BMPs to orient axons. Here, we show that BmprIb, and not BmprIa, is additionally required to control the rate of commissural axon extension. We have also determined the intracellular effector by which BmprIb regulates commissural axon growth. We show that BmprIb has a novel role modulating the activity of the actin-severing protein cofilin. These studies reveal the mechanistic differences used by distinct components of the canonical Bmpr complex to mediate the diverse activities of the BMPs. PMID:23250207

  18. CRTH2, a prostaglandin D2 receptor, mediates depression-related behavior in mice.

    PubMed

    Onaka, Yusuke; Shintani, Norihito; Nakazawa, Takanobu; Haba, Ryota; Ago, Yukio; Wang, Hyper; Kanoh, Takuya; Hayata-Takano, Atsuko; Hirai, Hiroyuki; Nagata, Kin-Ya; Nakamura, Masataka; Hashimoto, Ryota; Matsuda, Toshio; Waschek, James A; Kasai, Atsushi; Nagayasu, Kazuki; Baba, Akemichi; Hashimoto, Hitoshi

    2015-05-01

    Depression is a complex neuropsychiatric disorder with an unclear molecular etiology. Inflammatory cytokines and molecular intermediates (including prostaglandins) are suggested to be involved in depression; however, the roles of prostaglandins and their respective receptors are largely unknown in depression. Using genetic and pharmacological approaches, we show here that chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a second receptor for prostaglandin D2 (PGD2), mediates depression-related behavior in mice. CRTH2-deficient (CRTH2(-/-)) mice showed antidepressant-like activity in a chronic corticosterone treatment-induced depression. Consistent with this observation, the pharmacological inhibition of CRTH2 via the clinically available drug ramatroban also rescued abnormal social interaction and depression-related behavior in well-established models, including chronic corticosterone-, lipopolysaccharide-, and tumor-induced pathologically relevant depression models. Importantly, chronic stress via corticosterone treatment increased mRNA levels in PGD2-producing enzymes, such as cyclooxygenase-2 and lipocalin-type PGD2 synthase, in the brain. Furthermore, the activity of the hippocampal noradrenergic system but not the dopaminergic or serotonergic systems was increased in CRTH2(-/-) mice. Together with the observation that untreated CRTH2(-/-) mice showed antidepressant-like activity in the forced swim test, these results provide evidence that central CRTH2-mediated signaling is critically involved in depression-related behavior. PMID:25698598

  19. A putative octopamine/tyramine receptor mediating appetite in a hungry fly

    NASA Astrophysics Data System (ADS)

    Ishida, Yuko; Ozaki, Mamiko

    2011-07-01

    In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.

  20. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

    PubMed Central

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G.; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J.; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M.; Kaczmarek, Leonard K.; Flavell, Richard A.

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  1. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  2. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea.

    PubMed

    De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R

    2016-01-01

    While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (<24 h) phase following treatment, the anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments.

  3. P2 purinoceptor saturation by adenosine triphosphate impairs renal autoregulation in dogs.

    PubMed

    Majid, D S; Inscho, E W; Navar, L G

    1999-03-01

    Recent studies have suggested a role for P2 purinoceptors on vascular smooth muscle cells in the mechanism of renal autoregulation. Experiments were performed in anesthetized dogs (n = 9) to examine renal blood flow (RBF) autoregulatory efficiency before and after saturation of P2 purinoceptors with acute intra-arterial administration of ATP (1 mg/kg per min). Dogs were pretreated with the nitric oxide synthase inhibitor nitro-L-arginine (NLA) (50 microg/kg per min), to avoid endothelial P2 receptor-mediated effects on nitric oxide release caused by the intra-arterial ATP infusions. NLA treatment decreased RBF (5.3+/-0.3 to 3.6+/-0.2 ml/min per g) and sodium excretion (3.6+/-0.4 to 0.9+/-0.2 ml/min per g) without producing significant changes in GFR (0.92+/-0.04 to 0.90+/-0.06 ml/min per g) or RBF autoregulatory efficiency. ATP administration to NLA-treated dogs resulted in further decreases in RBF (2.8+/-0.2 ml/min per g), GFR (0.58+/-0.05 ml/min per g), and sodium excretion (0.6+/-0.2 micromol/min per g). In addition, there was marked impairment of RBF autoregulatory efficiency during ATP infusion. The slopes of the arterial pressure-blood flow relationships at renal arterial pressures of >75 mmHg were significantly altered, from 0.003+/-0.001 to 0.2+/-0.002 ml/min per g per mmHg. Discontinuation of ATP infusion restored RBF autoregulatory efficiency. Norepinephrine (5 microg/kg per min) administration in these NLA-treated dogs decreased RBF (2.5+/-0.3 ml/min per g; n = 4) to a similar extent, compared with ATP, but did not impair RBF autoregulation. These results support the hypothesis that P2 purinoceptors may be involved in mediating autoregulatory adjustments in renal vascular resistance. PMID:10073599

  4. Ligand-Specific Transcriptional Mechanisms Underlie Aryl Hydrocarbon Receptor-Mediated Developmental Toxicity of Oxygenated PAHs

    PubMed Central

    Goodale, B. C.; La Du, J.; Tilton, S. C.; Sullivan, C. M.; Bisson, W. H.; Waters, K. M.; Tanguay, R. L.

    2015-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds. PMID:26141390

  5. Adolescent chronic mild stress alters hippocampal CB1 receptor-mediated excitatory neurotransmission and plasticity.

    PubMed

    Reich, C G; Mihalik, G R; Iskander, A N; Seckler, J C; Weiss, M S

    2013-12-01

    Endocannabinoids (eCBs) are involved in the stress response and alterations in eCB signaling may contribute to the etiology of mood disorders. Exposure to chronic mild stress (CMS), a model of depression, produces downregulation of the cannabinoid 1 (CB1) receptor in the hippocampus of male rats. However, it is unknown how this stress-induced change in CB1 levels affects eCB-mediated neurotransmission. In vitro, field potential recordings from CMS-exposed (21-days) rats were performed to assess the effects of stress on eCB-regulated glutamatergic neurotransmission in/on hippocampal area CA1. We observed that application of the CB1 agonist, WIN 55,212-5 (1 μM), in stress animals resulted in a ∼135% increase in excitatory neurotransmission, whereas CB1 activation in non-stress animals leads to a ∼30% decrease. However, during blockade of GABA(A) neurotransmission with picrotoxin, CB1 activation yielded a ∼35% decrease in stress animals. These findings indicate that CMS does not directly affect glutamatergic neurotransmission. Rather, CMS sensitizes CB1 function on GABAergic terminals, leading to less inhibition and an increase in excitatory neurotransmission. This finding is reinforced in that induction of weak long-term-potentiation (LTP) is enhanced in CMS-exposed animals compared to controls and this enhancement is CB1-dependent. Lastly, we observed that the LTP-blocking property of WIN 55,212-5 shifts from being glutamate-dependent in non-stress animals to being GABA-dependent in stress animals. These results effectively demonstrate that CMS significantly alters hippocampal eCB-mediated neurotransmission and synaptic plasticity.

  6. Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells

    PubMed Central

    Liu, Hongguang; Singla, Amika; Ao, Mei; Gill, Ravinder K; Venkatasubramanian, Jayashree; Rao, Mrinalini C; Alrefai, Waddah A; Dudeja, Pradeep K

    2011-01-01

    Abstract High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT-secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT-PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (ISC) in a dose-dependent manner that was blocked by 1 μM of CTR antagonist, CT8–32. CT-induced ISC was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR127inh. Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca2+ chelator, bis(2-aminophenoxy)ethane tetraacetic acid-acetoxymethyl (BAPTA-AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited ISC induced by CT (∼32–58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA- and Ca2+-dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT-induced diarrhoea. PMID:21251218

  7. NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats

    PubMed Central

    Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.

    2010-01-01

    Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague–Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.− production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.− production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.− generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975–986. PMID:20406136

  8. NMDA Receptors Mediate Stimulus-Timing-Dependent Plasticity and Neural Synchrony in the Dorsal Cochlear Nucleus

    PubMed Central

    Stefanescu, Roxana A.; Shore, Susan E.

    2015-01-01

    Auditory information relayed by auditory nerve fibers and somatosensory information relayed by granule cell parallel fibers converge on the fusiform cells (FCs) of the dorsal cochlear nucleus, the first brain station of the auditory pathway. In vitro, parallel fiber synapses on FCs exhibit spike-timing-dependent plasticity with Hebbian learning rules, partially mediated by the NMDA receptor (NMDAr). Well-timed bimodal auditory-somatosensory stimulation, in vivo equivalent of spike-timing-dependent plasticity, can induce stimulus-timing-dependent plasticity (StTDP) of the FCs spontaneous and tone-evoked firing rates. In healthy guinea pigs, the resulting distribution of StTDP learning rules across a FC neural population is dominated by a Hebbian profile while anti-Hebbian, suppressive and enhancing LRs are less frequent. In this study, we investigate in vivo, the NMDAr contribution to FC baseline activity and long term plasticity. We find that blocking the NMDAr decreases the synchronization of FC- spontaneous activity and mediates differential modulation of FC rate-level functions such that low, and high threshold units are more likely to increase, and decrease, respectively, their maximum amplitudes. Three significant alterations in mean learning-rule profiles were identified: transitions from an initial Hebbian profile towards (1) an anti-Hebbian; (2) a suppressive profile; and (3) transitions from an anti-Hebbian to a Hebbian profile. FC units preserving their learning rules showed instead, NMDAr-dependent plasticity to unimodal acoustic stimulation, with persistent depression of tone-evoked responses changing to persistent enhancement following the NMDAr antagonist. These results reveal a crucial role of the NMDAr in mediating FC baseline activity and long-term plasticity which have important implications for signal processing and auditory pathologies related to maladaptive plasticity of dorsal cochlear nucleus circuitry. PMID:26622224

  9. Ligand-specific transcriptional mechanisms underlie aryl hydrocarbon receptor-mediated developmental toxicity of oxygenated PAHs

    SciTech Connect

    Goodale, B. C.; La Du, J.; Tilton, S. C.; Sullivan, C. M.; Bisson, W. H.; Waters, K. M.; Tanguay, R. L.

    2015-07-03

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Furthermore, identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds.

  10. Ligand-specific transcriptional mechanisms underlie aryl hydrocarbon receptor-mediated developmental toxicity of oxygenated PAHs

    DOE PAGES

    Goodale, B. C.; Geisel School of Medicine at Dartmouth, Hanover, NH; La Du, J.; Tilton, S. C.; Pacific Northwest National Lab.; Sullivan, C. M.; Bisson, W. H.; Waters, K. M.; Tanguay, R. L.

    2015-07-03

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, butmore » only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Furthermore, identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds.« less

  11. Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

    PubMed

    Zheng, Yu; Song, Xiangrong; Darby, Michael; Liang, Yufeng; He, Ling; Cai, Zheng; Chen, Qiuhong; Bi, Yueqi; Yang, Xiaojuan; Xu, Jiapeng; Li, Yuanbo; Sun, Yiyi; Lee, Robert J; Hou, Shixiang

    2010-01-01

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

  12. Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines.

    PubMed

    Kouchi, Zen

    2015-08-21

    Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.

  13. Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

    PubMed Central

    de Torre-Minguela, Carlos; Barberà-Cremades, Maria; Gómez, Ana I.; Martín-Sánchez, Fátima; Pelegrín, Pablo

    2016-01-01

    The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P2X7R stimulation goes beyond caspase-1 secretome. The characterization of P2X7R-secretome reveals a new function of this receptor through a fine-tuning of protein release. We found that P2X7R stimulation in macrophages is able to release potent anti-inflammatory proteins, such as Annexin A1, independently of their polarization state suggesting for first time a potential role for P2X7R during resolution of the inflammation and not linked to the release of pro-inflammatory cytokines. These results are of prime importance for the development of therapeutics targeting P2X7R. PMID:26935289

  14. Chemotherapy-induced uridine diphosphate release promotes breast cancer metastasis through P2Y6 activation

    PubMed Central

    Ma, Xiaobin; Pan, Xinhua; Wei, Yinglei; Tan, Binhe; Yang, Linli; Ren, Hua; Qian, Min; Du, Bing

    2016-01-01

    Although purinergic signaling is important in regulation of immune responses, the therapeutic potential of it in the tumor microenvironment is little defined. In this study, we demonstrate that UDP/P2Y6 signaling facilitates breast cancer metastasis both in vitro and in vivo. We found that P2Y6 is not only aberrantly expressed and mutated in most tumor types, but also highly correlated with poor prognosis in breast cancer patients. Furthermore, the migration and invasion of breast cancer cells was obviously increased by UDP and blocked by P2Y6 specific inhibitor MRS2578 and P2Y6 shRNA. Similar results was also found in breast cancer cell metastasis mouse model. Interestingly, the endogenous agonist UDP was released significantly by doxorubicin treated cells. In addition, the expression and enzyme activity of MMP-9 were both promoted by UDP and inhibited by MRS2578 or P2Y6 shRNA. Furthermore, UDP-induced cell invasion was blocked by an MMP-9 inhibitor. Mechanistically, the MAPKs and NF-κB signaling pathways, known to be involved in regulation of MMP-9 expression, were both activated by UDP. Taken together, our study reveals a relationship between extracellular danger signals and breast cancer metastasis, which suggests the potential therapeutic significance of UDP/P2Y6 signaling in cancer therapy. PMID:27074554

  15. Kappa Opioid Receptor-Mediated Disruption of Novel Object Recognition: Relevance for Psychostimulant Treatment

    PubMed Central

    Paris, Jason J.; Reilley, Kate J.; McLaughlin, Jay P.

    2012-01-01

    Kappa opioid receptor (KOR) agonists are potentially valuable as therapeutics for the treatment of psychostimulant reward as they suppress dopamine signaling in reward circuitry to repress drug seeking behavior. However, KOR agonists are also associated with sedation and cognitive dysfunction. The extent to which learning and memory disruption or hypolocomotion underlie KOR agonists’ role in counteracting the rewarding effects of psychostimulants is of interest. C57BL/6J mice were pretreated with vehicle (saline, 0.9%), the KOR agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), or the peripherally-restricted agonist D-Phe-D-Phe-D-lle-D-Arg- NH2 (ffir-NH2), through central (i.c.v.) or peripheral (i.p.) routes of administration. Locomotor activity was assessed via activity monitoring chambers and rotorod. Cognitive performance was assessed in a novel object recognition task. Prolonged hypolocomotion was observed following administration of 1.0 and 10.0, but not 0.3 mg/kg U50,488. Central, but not peripheral, administration of ffir-NH2 (a KOR agonist that does not cross the blood-brain barrier) also reduced motor behavior. Systemic pretreatment with the low dose of U50,488 (0.3 mg/kg, i.p.) significantly impaired performance in the novel object recognition task. Likewise, ffir-NH2 significantly reduced novel object recognition after central (i.c.v.), but not peripheral (i.p.), administration. U50,488- and ffir-NH2-mediated deficits in novel object recognition were prevented by pretreatment with KOR antagonists. Cocaine-induced conditioned place preference was subsequently assessed and was reduced by pretreatment with U50,488 (0.3 mg/kg, i.p.). Together, these results suggest that the activation of centrally-located kappa opioid receptors may induce cognitive and mnemonic disruption independent of hypolocomotor effects which may contribute to the KOR-mediated suppression of psychostimulant reward. PMID:22900234

  16. The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

    PubMed

    Kim, Eun-A; Cho, Chang Hun; Kim, Jiae; Hahn, Hoh-Gyu; Choi, Soo Young; Yang, Seung-Ju; Cho, Sung-Woo

    2015-12-01

    Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia.

  17. P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation

    PubMed Central

    Buvinic, Sonja; Poblete, M Inés; Donoso, M Verónica; Delpiano, Ana María; Briones, René; Miranda, Ramiro; Huidobro-Toro, J Pablo

    2006-01-01

    The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y1R and P2Y2R revealed a 6- to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y2R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y1R and P2Y2R were detected by RT-PCR and the mRNA coding for the P2Y4R, P2Y6R and P2Y11R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y1R and P2Y2R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration-dependent manner by 10–100 nm indomethacin or 10 nm GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y1R antagonist, reduced the 2-MeSADP- but not the UTP-evoked contractions. Perfusion of cotyledons with 2-MeSADP or UTP evoked concentration-dependent reductions in perfusion pressure mediated by the NO–cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2-MeSADP but not by UTP. In summary, P2Y1R and P2Y2R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses. PMID:16543271

  18. P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation.

    PubMed

    Buvinic, Sonja; Poblete, M Inés; Donoso, M Verónica; Delpiano, Ana María; Briones, René; Miranda, Ramiro; Huidobro-Toro, J Pablo

    2006-06-01

    The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y(1)R and P2Y(2)R revealed a 6- to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y(2)R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y(1)R and P2Y(2)R were detected by RT-PCR and the mRNA coding for the P2Y(4)R, P2Y(6)R and P2Y(11)R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y(1)R and P2Y(2)R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration-dependent manner by 10-100 nm indomethacin or 10 nm GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y(1)R antagonist, reduced the 2-MeSADP- but not the UTP-evoked contractions. Perfusion of cotyledons with 2-MeSADP or UTP evoked concentration-dependent reductions in perfusion pressure mediated by the NO-cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2-MeSADP but not by UTP. In summary, P2Y(1)R and P2Y(2)R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses.

  19. Regulation of rat cortical 5-hydroxytryptamine2A-receptor mediated electrophysiological responses by repeated daily treatment with electroconvulsive shock or imipramine

    PubMed Central

    Marek, Gerard J.

    2008-01-01

    Down-regulation of 5-hydroxytryptamine2A (5-HT2A) receptors has been a consistent effect induced by most antidepressant drugs. In contrast, electroconvulsive shock (ECS) up-regulates the number of 5-HT2A receptor binding sites. However, the effects of antidepressants on 5-HT2A receptor-mediated responses on identified cells of the cerebral cortex has not been examined. The purpose of the present study was to compare the effects of the tricyclic antidepressant imipramine and ECS on 5-HT2A receptor-mediated electrophysiological responses involving glutamatergic and GABAergic neurotransmission in the rat medial prefrontal cortex (mPFC) and piriform cortex, respectively. The electrophysiological effects of activating 5-HT2A receptors was consistent with 5-HT2A receptor binding regulation for imipramine and ECS except for the mPFC where chronic ECS decreased the potency of 5-HT at a 5-HT2A receptor-mediated response. These findings are consistent with the general hypothesis that chronic antidepressant treatments shift the balance of serotonergic neurotransmission towards inhibitory effects in the cortex. PMID:18294819

  20. Regulation of rat cortical 5-hydroxytryptamine2A receptor-mediated electrophysiological responses by repeated daily treatment with electroconvulsive shock or imipramine.

    PubMed

    Marek, Gerard J

    2008-07-01

    Down-regulation of 5-hydroxytryptamine(2A) (5-HT(2A)) receptors has been a consistent effect induced by most antidepressant drugs. In contrast, electroconvulsive shock (ECS) up-regulates the number of 5-HT(2A) receptor binding sites. However, the effects of antidepressants on 5-HT(2A) receptor-mediated responses on identified cells of the cerebral cortex have not been examined. The purpose of the present study was to compare the effects of the tricyclic antidepressant imipramine and ECS on 5-HT(2A) receptor-mediated electrophysiological responses involving glutamatergic and GABAergic neurotransmission in the rat medial prefrontal cortex (mPFC) and piriform cortex, respectively. The electrophysiological effects of activating 5-HT(2A) receptors were consistent with 5-HT(2A) receptor binding regulation for imipramine and ECS except for the mPFC where chronic ECS decreased the potency of 5-HT at a 5-HT(2A) receptor-mediated response. These findings are consistent with the general hypothesis that chronic antidepressant treatments shift the balance of serotonergic neurotransmission towards inhibitory effects in the cortex.

  1. Diurnal dynamic behavior of microglia in response to infected bacteria through the UDP-P2Y6 receptor system

    PubMed Central

    Takayama, Fumiko; Hayashi, Yoshinori; Wu, Zhou; Liu, Yicong; Nakanishi, Hiroshi

    2016-01-01

    It has long been believed that microglia morphologically transform into the activated state by retracting their long processes and consuming pathogens when bacteria infect into the brain parenchyma. In the present study, however, we showed for the first time that murine cortical microglia extend their processes towards focally injected Porphyromonas gingivalis. This P. gingivalis-induced microglial process extension was significantly increased during the light (sleeping) phase than the dark (waking) phase. In contrast, focally injected ATP-induced microglial process extension was significantly increased during the dark phase than the light phase. Furthermore, in contrast to the P2Y12 receptor-mediated mechanism of ATP-induced microglial process extension, the P. gingivalis-mediated microglial process extension was mediated by P2Y6 receptors. The infection of bacteria such as P. gingivalis to the brain parenchyma may induce the secretion of UDP from microglia at the site of infection, which in turn induces the process extension of the neighboring microglia. PMID:27445174

  2. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

  3. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways. PMID:11389765

  4. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    SciTech Connect

    Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  5. 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

  6. Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

    PubMed

    Ye, Zhaoyang; Cheng, Kun; Guntaka, Ramareddy V; Mahato, Ram I

    2006-01-01

    Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

  7. Ca(2+)-independent F-actin assembly and disassembly during Fc receptor- mediated phagocytosis in mouse macrophages

    PubMed Central

    1991-01-01

    Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F- actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F- actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i. PMID:2026648

  8. Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the Ah phenotype of lymphoid tissue.

    PubMed

    Silkworth, J B; Antrim, L A; Sack, G

    1986-12-01

    Halogenated aromatic hydrocarbons act through the aromatic hydrocarbon (Ah) receptor in mice to produce a series of toxic effects of the immune system. The receptor protein is a product of the Ah gene locus. Ah responsive (Ahb/Ahb) mice express a high affinity receptor in both lymphoid and nonlymphoid tissues whereas nonresponsive Ahd/Ahd mice express a poor affinity receptor. To determine the role of the Ah receptor of lymphoid tissue relative to that of nonlymphoid tissue in the induction of immune impairment, bone marrow was used to reconstitute lethally irradiated mice of the same or opposite Ah phenotype. All mice were given 3,3',4,4'-tetrachlorobiphenyl (35 and 350 mumol/kg) ip 2 days before immunization with sheep erythrocytes (SRBC). The immune response to this T dependent antigen and organ weights were determined 5 or 7 days later in normal or chimeric mice, respectively. Monoclonal Lyt 1.1 and Lyt 1.2 antibodies were used to establish the origin of the cells which repopulated the chimeric thymuses. The immune responses of both BALB/cBy (Ahb/Ahb) and the BALB/cBy X DBA/2 hybrid, CByD2F1 (Ahb/Ahd), were significantly suppressed but DBA/2 mice were unaffected. The immune responses of chimeric BALB/cBy----BALB/cBy and BALB/cBy----DBA/2 (donor----recipient) mice were also significantly suppressed and thymic atrophy was observed in both cases. The serum anti-SRBC antibody titers of DBA/2----BALB/cBy chimeras were also significantly decreased although not to the same extent as in BALB/cBy----DBA/2 mice. Chimeric DBA/2----DBA/2 mice were not affected. These results indicate that the sensitivity to Ah receptor mediated suppression of the antibody response is primarily determined by the Ah phenotype of the lymphoid tissue.

  9. Modulation of the input–output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons

    PubMed Central

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-01-01

    The neuronal input–output function depends on recruitment threshold and gain of the firing frequency–current (f–I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input–output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f–I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f–I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation. PMID:25194049

  10. Gonadotrophin-releasing activity of neurohypophysial hormones: II. The pituitary oxytocin receptor mediating gonadotrophin release differs from that of corticotrophs.

    PubMed

    Evans, J J; Catt, K J

    1989-07-01

    Neurohypophysial hormones stimulate gonadotrophin release from dispersed rat anterior pituitary cells in vitro, acting through receptors distinct from those which mediate the secretory response to gonadotrophin-releasing hormone (GnRH). The LH response to oxytocin was not affected by the presence of the phosphodiesterase inhibitor, methyl isobutylxanthine, but was diminished in the absence of extracellular calcium and was progressively increased as the calcium concentration in the medium was raised to normal. In addition, the calcium channel antagonist, nifedipine, suppressed oxytocin-stimulated secretion of LH. It is likely that the mechanisms of LH release induced by GnRH and neurohypophysial hormones are similar, although stimulation of gonadotrophin secretion is mediated by separate receptor systems. Oxytocin was more active than vasopressin in releasing LH, but less active in releasing ACTH. The highly selective oxytocin agonist, [Thr4,Gly7]oxytocin, elicited concentration-dependent secretion of LH but had little effect on corticotrophin secretion. The neurohypophysial hormone antagonist analogues, [d(CH2)5Tyr(Me)2]vasopressin, [d(CH2)5Tyr(Me)2,Orn8]vasotocin and [d(CH2)5D-Tyr(Et)2Val4,Cit8]vasopressin, inhibited the LH response to both oxytocin and vasopressin. However, [d(CH2)5Tyr(Me)2]vasopressin was much less effective in inhibiting the ACTH response to the neurohypophysial hormones, and [d(CH2)5Tyr-(Me)2,Orn8]vasotocin and [d(CH2)5D-Tyr(Et)2,Val4,Cit8]vasopressin exhibited no inhibitory activity against ACTH release. Thus, agonist and antagonist analogues of neurohypophysial hormones display divergent activities with regard to LH and ACTH responses, and the neuropeptide receptor mediating gonadotroph activation is clearly different from that on the corticotroph. Whereas the corticotroph receptor is a vasopressin-type receptor an oxytocin-type receptor is responsible for gonadotrophin release by neurohypophysial hormones.

  11. Two types of functionally different GABAA receptors mediate GABA modulation of cholinergic transmission in cat terminal ileum.

    PubMed

    Radomirov, R; Pencheva, N

    1995-08-01

    1. The effects of GABA (1 microM-2 mM) on longitudinally or circularly oriented organ bath preparations of cat terminal ileum consisted of a relaxation phase with an inhibition of the rhythmic spontaneous phasic contractions, followed by a phase of contractions characterized by an elevation in basal tone and an increase in amplitude of the spontaneous phasic contractions. 2. Muscimol (100 microM), but not baclofen (100 microM), mimicked the relaxation phase of the response to applied GABA (100 microM) in all tissue preparations. In addition, muscimol induced a phase of contractile activity in the circular muscle layer whilst baclofen exerted a 'GABA-like' contractile effect on the longitudinal muscle layer. Bicuculline (30 microM) or picrotoxinin (30 microM) antagonized the GABA- or muscimol-induced relaxations in all preparations and decreased the GABA- but not the baclofen-induced contractions of the longitudinal muscle layer. 3. Tetrodotoxin (0.5 microM) or atropine (0.1 microM) prevented the bicuculline-sensitive phases of the GABA or muscimol effects on both muscle layers but not the contractile effect of baclofen on the longitudinal muscle layer. 4. The bicuculline-sensitive phases of the GABA effect on both muscle layers were almost completely eliminated by 1 nM pirenzepine. At this concentration pirenzepine did not affect the electrically-evoked cholinergic twitch contractions or contractile responses to applied acetylcholine of both muscle layers. 5. During electrically-evoked cholinergic twitch contractions of both muscle layers, GABA (100 microM) had an inhibitory effect. The inhibition occurred in the presence of pirenzepine (1 nM) but not of bicuculline (30 microM). 6. It is suggested that two types of functionally different bicuculline-sensitive GABAA receptors mediate an exitatory presynaptic and an inhibitory prejunctional action of GABA on the cholinergic transmission in cat terminal ileum.

  12. Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

    PubMed

    Deli, M A; Abrahám, C S; Takahata, H; Katamine, S; Niwa, M

    2000-12-01

    1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

  13. Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

    PubMed Central

    Modun, Belinda; Evans, Robert W.; Joannou, Christopher L.; Williams, Paul

    1998-01-01

    Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and

  14. Early growth response-1 suppresses epidermal growth factor receptor-mediated airway hyperresponsiveness and lung remodeling in mice.

    PubMed

    Kramer, Elizabeth L; Mushaben, Elizabeth M; Pastura, Patricia A; Acciani, Thomas H; Deutsch, Gail H; Khurana Hershey, Gurjit K; Korfhagen, Thomas R; Hardie, William D; Whitsett, Jeffrey A; Le Cras, Timothy D

    2009-10-01

    Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.

  15. Role of various kinases in muscarinic M3 receptor-mediated contraction of longitudinal muscle of rat colon

    PubMed Central

    Anderson, Charles D.; Kendig, Derek M.; Al-Qudah, Mohammad; Mahavadi, Sunila; Murthy, Karnam S.; Grider, John R.

    2016-01-01

    The longitudinal muscle layer in gut is the functional opponent to the circular muscle layer during peristalsis. Differences in innervation of the layers allow for the contraction of one layer concurrently with the relaxation of the other, enabling the passage of gut contents in a controlled fashion. Differences in development have given the cells of the two layers differences in receptor populations, membrane lipid handling, and calcium handling profiles/behaviors. The contractile activity of the longitudinal muscle is largely mediated by cholinergic neural input from myenteric plexus. Activation of muscarinic receptors leads to rapid activation of several kinases including MLC kinase, ERK1/2, CaMKII and Rho kinase. Phosphorylation of myosin light chain (MLC20) by MLC kinase (MLCK) is a prerequisite for contraction in both circular and longitudinal muscle cells. In rat colonic longitudinal muscle strips, we measured muscarinic receptor-mediated contraction following incubation with kinase inhibitors. Basal tension was differentially regulated by Rho kinase, ERK1/2, CaMKII and CaMKK. Selective inhibitors of Rho kinase, ERK1/2, CaMKK/AMPK, and CaMKII each reduced carbachol-induced contraction in the innervated muscle strips. These inhibitors had no direct effect on MLCK activity. Thus unlike previously reported for isolated muscle cells where CaMKII and ERK1/2 are not involved in contraction, we conclude that the regulation of carbachol-induced contraction in innervated longitudinal muscle strips involves the interplay of Rho kinase, ERK1/2, CaMKK/AMPK, and CAMKII. PMID:25891767

  16. Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

    PubMed

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-11-15

    The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

  17. Role of leptin signaling in hemato-vascular development and niche function: Leptin receptor-mediated signaling regulates LT-HSC homeostasis in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homeostatic functioning of the cardiovascular and hematopoietic systems is known to be interdependent and strongly influenced by the microenvironment in which hemato-vascular cells develop and reside. The role of nutrition and metabolism as regulable and dynamic extracellular cues however, remains a...

  18. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    PubMed Central

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  19. Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation

    PubMed Central

    Riegel, Ann-Kathrin; Faigle, Marion; Zug, Stephanie; Rosenberger, Peter; Robaye, Bernard; Boeynaems, Jean-Marie

    2011-01-01

    During a systemic inflammatory response endothelial-expressed surface molecules have been strongly implicated in orchestrating immune responses. Previous studies have shown enhanced extracellular nucleotide release during acute inflammatory conditions. Therefore, we hypothesized that endothelial nucleotide receptors could play a role in vascular inflammation. To address this hypothesis, we performed screening experiments and exposed human microvascular endothelia to inflammatory stimuli, followed by measurements of P2Y or P2X transcriptional responses. These studies showed a selective induction of the P2Y6 receptor (> 4-fold at 24 hours). Moreover, studies that used real-time reverse transcription–polymerase chain reaction, Western blot analysis, or immunofluorescence confirmed time- and dose-dependent induction of P2Y6 with tumor necrosis factor α or Lipopolysaccharide (LPS) stimulation in vitro and in vivo. Studies that used MRS 2578 as P2Y6 receptor antagonist showed attenuated nuclear factor κB reporter activity and proinflammatory gene expression in human microvascular endothelial cells in vitro. Moreover, pharmacologic or genetic in vivo studies showed attenuated inflammatory responses in P2Y6−/− mice or after P2Y6 antagonist treatment during LPS-induced vascular inflammation. These studies show an important contribution of P2Y6 signaling in enhancing vascular inflammation during systemic LPS challenge and implicate the P2Y6 receptor as a therapeutic target during systemic inflammatory responses. PMID:21173118

  20. Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

    PubMed

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  1. Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism.

    PubMed

    Kimball, B C; Yule, D I; Mulholland, M W

    1996-04-01

    In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.

  2. The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells

    PubMed Central

    Sathanoori, Ramasri; Swärd, Karl; Olde, Björn; Erlinge, David

    2015-01-01

    Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase

  3. Receptor-mediated mitophagy.

    PubMed

    Yamaguchi, Osamu; Murakawa, Tomokazu; Nishida, Kazuhiko; Otsu, Kinya

    2016-06-01

    Mitochondria are essential organelles that supply ATP through oxidative phosphorylation to maintain cellular homeostasis. Extrinsic or intrinsic agents can impair mitochondria, and these impaired mitochondria can generate reactive oxygen species (ROS) as byproducts, inducing cellular damage and cell death. The quality control of mitochondria is essential for the maintenance of normal cellular functions, particularly in cardiomyocytes, because they are terminally differentiated. Accumulation of damaged mitochondria is characteristic of various diseases, including heart failure, neurodegenerative disease, and aging-related diseases. Mitochondria are generally degraded through autophagy, an intracellular degradation system that is conserved from yeast to mammals. Autophagy is thought to be a nonselective degradation process in which cytoplasmic proteins and organelles are engulfed by isolation membrane to form autophagosomes in eukaryotic cells. However, recent studies have described the process of selective autophagy, which targets specific proteins or organelles such as mitochondria. Mitochondria-specific autophagy is called mitophagy. Dysregulation of mitophagy is implicated in the development of chronic diseases including neurodegenerative diseases, metabolic diseases, and heart failure. In this review, we discuss recent progress in research on mitophagy receptors. PMID:27021519

  4. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    SciTech Connect

    Mistafa, Oras; Hoegberg, Johan; Stenius, Ulla

    2008-01-04

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells.

  5. Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

    PubMed

    Corso, Lucia; Cavallero, Anna; Baroni, Debora; Garbati, Patrizia; Prestipino, Gianfranco; Bisti, Silvia; Nobile, Mario; Picco, Cristiana

    2016-03-01

    P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases. PMID:26739703

  6. Prolonged GABA(B) receptor-mediated synaptic inhibition in the cat spinal cord: an in vivo study.

    PubMed

    Curtis, D R; Lacey, G

    1998-08-01

    methochloride. Intravenously administered bicuculline hydrochloride, however, had little or no effect on the inhibition of reflexes following continuous flexor-nerve stimulation. These observations are discussed in the context of possible intraspinal pathways and pre- and postsynaptic mechanisms for GABA(A) and GABA(B) receptor-mediated inhibition of the monosynaptic excitation of spinal motoneurones and of the functional significance of central GABA(B) receptor-associated inhibitory processes, given the relatively minimal effects on motor activity and behaviour produced by baclofen antagonists that penetrate the mammalian blood-brain barrier.

  7. Oligodendrocytes Are Targets of HIV-1 Tat: NMDA and AMPA Receptor-Mediated Effects on Survival and Development

    PubMed Central

    Zou, Shiping; Fuss, Babette; Fitting, Sylvia; Hahn, Yun Kyung; Hauser, Kurt F.

    2015-01-01

    Myelin pallor in HIV+ individuals can occur very early during the disease process. While myelin damage might partly originate from HIV-induced vascular changes, the timing suggests that myelin and/or oligodendrocytes (OLs) may be directly affected. Histological (Golgi–Kopsch, electron microscopy) and biochemical studies have revealed an increased occurrence of abnormal OL/myelin morphology and dysregulated myelin protein expression in transgenic mice expressing the HIV-1 transactivator of transcription (Tat) protein. This suggests that viral proteins by themselves might cause OL injury. Since Tat interacts with NMDARs, we hypothesized that activation of NMDARs and subsequent disruption of cytoplasmic Ca2+ ([Ca2+]i) homeostasis might be one cause of white matter injury after HIV infection. In culture, HIV-1 Tat caused concentration-dependent death of immature OLs, while more mature OLs remained alive but had reduced myelin-like membranes. Tat also induced [Ca2+]i increases and Thr-287 autophosphorylation of Ca2+/calmodulin-dependent protein kinase II β (CaMKIIβ) in OLs. Tat-induced [Ca2+]i was attenuated by the NMDAR antagonist MK801, and also by the AMPA/kainate receptor antagonist CNQX. Importantly, both MK801 and CNQX blocked Tat-induced death of immature OLs, but only MK801 reversed Tat effects on myelin-like membranes. These results suggest that OLs can be direct targets of HIV proteins released from infected cells. Although viability and membrane production are both affected by glutamatergic receptor-mediated Ca2+ influx, and possibly the ensuing CaMKIIβ activation, the roles of AMPARs and NMDARs appear to be different and dependent on the stage of OL differentiation. SIGNIFICANCE STATEMENT Over 33 million individuals are currently infected by HIV. Among these individuals, ∼60% develop HIV-associated neurocognitive disorders. Myelin damage and white matter injury have been frequently reported in HIV patients but not extensively studied. Clinical studies

  8. α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

    PubMed

    Inden, Masatoshi; Takata, Kazuyuki; Yanagisawa, Daijiro; Ashihara, Eishi; Tooyama, Ikuo; Shimohama, Shun; Kitamura, Yoshihisa

    2016-03-01

    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no

  9. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  10. Spin reorientation transition in ultrathin Co film on InP(2x4) reconstructed surface

    SciTech Connect

    Park, Yong-Sung; Jeong, Jong-Ryul; Shin, Sung-Chul

    2005-05-15

    We have investigated magnetic properties of monolayer (ML)-thickness Co film deposited on InP(2x4) reconstructed surface using in situ surface magneto-optical Kerr effects (SMOKE) measurement system. InP(2x4) reconstructed surface, obtained by several cycles of sputtering-and-annealing process, was confirmed by reflection high energy electron diffraction (RHEED) and scanning tunneling microscopy (STM) measurements. Co film grown on InP(2x4) reconstructed surface shows three distinguishable thickness regions which have different magnetic properties, depending on Co film thickness. In the Co film thickness region smaller than 7 ML, no SMOKE signal was detected. In the thickness region between 8 ML and 15 ML, both longitudinal and polar Kerr hysteresis loops were observed. In the film thickness larger than 16 ML, only longitudinal SMOKE signal without polar signal was detected.

  11. P2CS: updates of the prokaryotic two-component systems database

    PubMed Central

    Ortet, Philippe; Whitworth, David E.; Santaella, Catherine; Achouak, Wafa; Barakat, Mohamed

    2015-01-01

    The P2CS database (http://www.p2cs.org/) is a comprehensive resource for the analysis of Prokaryotic Two-Component Systems (TCSs). TCSs are comprised of a receptor histidine kinase (HK) and a partner response regulator (RR) and control important prokaryotic behaviors. The latest incarnation of P2CS includes 164 651 TCS proteins, from 2758 sequenced prokaryotic genomes. Several important new features have been added to P2CS since it was last described. Users can search P2CS via BLAST, adding hits to their cart, and homologous proteins can be aligned using MUSCLE and viewed using Jalview within P2CS. P2CS also provides phylogenetic trees based on the conserved signaling domains of the RRs and HKs from entire genomes. HK and RR trees are annotated with gene organization and domain architecture, providing insights into the evolutionary origin of the contemporary gene set. The majority of TCSs are encoded by adjacent HK and RR genes, however, ‘orphan’ unpaired TCS genes are also abundant and identifying their partner proteins is challenging. P2CS now provides paired HK and RR trees with proteins from the same genetic locus indicated. This allows the appraisal of evolutionary relationships across entire TCSs and in some cases the identification of candidate partners for orphan TCS proteins. PMID:25324303

  12. Role of purinergic P2X receptors in the control of liver homeostasis.

    PubMed

    Fausther, Michel; Gonzales, Emmanuel; Dranoff, Jonathan A

    2012-05-01

    It is now accepted that extracellular ATP and other nucleotides are potent signaling molecules, akin to neurotransmitters, hormones and lipid mediators. In the liver, several clues support a significant role for extracellular ATP-induced signaling pathways in the control of tissue homeostasis. First, ATP and other nucleotides are physiologically detected in extracellular fluids within the liver, including sinusoidal blood and intraductular bile, in various mammalian species including human and rodents. Moreover, finely tuned mechanisms of ATP release by different liver cell types have been described, under physiological cellular changes. In addition, most hepatic cells constitutively express, at the membrane level, several ATP-metabolizing ectoenzymes and ATP-sensitive receptors that modulate and transduce these mediator signals respectively. Finally, hepatic cells also express numerous membrane transporters that actively contribute to purinergic salvage pathways. Once released in the extracellular medium, unmetabolised ATP molecules can bind to purinergic P2X and P2Y receptors, and subsequently trigger various intracellular signal transduction pathways collectively referred to as purinergic signaling. In the liver, purinergic signaling has been shown to regulate key basic cellular functions, such as glucose/lipid metabolism, protein synthesis and ionic secretion, and homeostatic processes, such as cell cycle, inflammatory response and immunity. Whilst the functional relevance of P2Y receptors in liver physiology has been well documented, limited information is available regarding the potential role of hepatic P2X receptors in the modulation of liver homeostasis.

  13. Signaling Pathways Used by Ergot Alkaloids to Inhibit Bovine Sperm Motility

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ergot alkaloids exert their toxic or pharmaceutical effects through membrane receptor-mediated activities. This study investigated the signaling pathways involved in the in vitro inhibitory effects of both ergotamine (ET) and dihydroergotamine (DEHT) on bovine sperm motility using specific inhibitor...

  14. Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity.

    PubMed

    Chen, H S; Pellegrini, J W; Aggarwal, S K; Lei, S Z; Warach, S; Jensen, F E; Lipton, S A

    1992-11-01

    Excessive activation of NMDA receptors is thought to mediate the calcium-dependent neurotoxicity associated with hypoxic-ischemic brain injury, trauma, epilepsy, and several neurodegenerative diseases. For this reason, various NMDA antagonists have been investigated for their therapeutic potential in these diseases, but heretofore none have proven to be both effective and safe. In the present study, memantine, an adamantane derivative similar to the antiviral drug amantadine, is shown to block the channels activated by NMDA receptor stimulation. From whole-cell and single-channel recording experiments, the mechanism of action of memantine is deduced to be open-channel block, similar to MK-801; however, unlike MK-801, memantine is well tolerated clinically. Compared to MK-801, memantine's safety may be related to its faster kinetics of action with rapid blocking and unblocking rates at low micromolar concentrations. Furthermore, at these levels memantine is an uncompetitive antagonist and should theoretically allow near-normal physiological NMDA activity throughout the brain even in the face of pathologically high focal concentrations of glutamate. These pharmacological properties confer upon memantine a therapeutic advantage against NMDA receptor-mediated neurotoxicity with few side effects compared with other organic NMDA open-channel blockers. Moreover, memantine is increasingly effective against escalating levels of glutamate, such as those observed during a stroke. Low micromolar concentrations of memantine, levels known to be tolerated by patients receiving the drug for the treatment of Parkinson's disease, prevent NMDA receptor-mediated neurotoxicity in cultures of rat cortical and retinal ganglion cell neurons; memantine also appears to be both safe and effective in a rat stroke model. These results suggest that memantine has considerable therapeutic potential for the myriad of clinical entities associated with NMDA receptor-mediated neurotoxicity.

  15. Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats.

    PubMed

    Hannon, J P; Tigani, B; Wolber, C; Williams, I; Mazzoni, L; Howes, C; Fozard, J R

    2002-02-01

    The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response. Like adenosine, the broad-spectrum adenosine receptor agonist, NECA, induced dose-related bronchoconstriction in actively sensitized, OA-challenged animals. In contrast, CPA, CGS 21680 and 2-Cl-IB-MECA, agonists selective for A(1) A(2A) and A(3) receptors, respectively, induced no, or minimal, bronchoconstriction. Neither the selective A(1) receptor antagonist, DPCPX, nor the selective A(2A) receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine. MRS 1754, which has similar affinity for rat A(2B) and A(1) receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A(1) receptor-mediated bradycardia induced by NECA. 8-SPT and CGS 15943, antagonists at A(1), A(2A), and A(2B) but not A(3) receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the K(B) value at the rat A(2B) receptor, respectively. Adenosine and NECA, but not CPA, CGS 21680 or 2-Cl-IB-MECA, induced contraction of parenchymal strip preparations from actively sensitized OA-challenged animals. Responses to adenosine could not be antagonized by 8-SPT or MRS 1754 at concentrations >50 times their affinities at the rat A(2B) receptor. The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

  16. Development of drug loaded nanoparticles for tumor targeting. Part 2: Enhancement of tumor penetration through receptor mediated transcytosis in 3D tumor models

    NASA Astrophysics Data System (ADS)

    El-Dakdouki, Mohammad H.; Puré, Ellen; Huang, Xuefei

    2013-04-01

    We report that receptor mediated transcytosis can be utilized to facilitate tumor penetration by drug loaded nanoparticles (NPs). We synthesized hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44 expressed on the cancer cell surface. Although prior studies have primarily focused on CD44 mediated endocytosis to facilitate cellular uptake of HA-NPs by cancer cells, we discovered that, once internalized, the HA-SNPs could be transported out of the cells with their cargo. The exported NPs could be taken up by neighboring cells. This enabled the HA-SNPs to penetrate deeper inside tumors and reach a much greater number of tumor cells in 3D tumor models, presumably through tandem cycles of CD44 mediated endocytosis and exocytosis. When doxorubicin (DOX) was loaded onto the NPs, better penetration of multilayered tumor cells was observed with much improved cytotoxicities against both drug sensitive and drug resistant cancer spheroids compared to the free drug. Thus, targeting receptors such as CD44 that can readily undergo recycling between the cell surface and interior of the cells can become a useful strategy to enhance the tumor penetration potential of NPs and the efficiency of drug delivery through receptor mediated transcytosis.We report that receptor mediated transcytosis can be utilized to facilitate tumor penetration by drug loaded nanoparticles (NPs). We synthesized hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44 expressed on the cancer cell surface. Although prior studies have primarily focused on CD44 mediated endocytosis to facilitate cellular uptake of HA-NPs by cancer cells, we discovered that, once internalized, the HA-SNPs could be transported out of the cells with their cargo. The exported NPs could be taken up by neighboring cells. This enabled the HA-SNPs to penetrate deeper inside tumors and reach a much greater number of tumor cells in 3D tumor

  17. Non-ionotropic signaling by the NMDA receptor: controversy and opportunity.

    PubMed

    Gray, John A; Zito, Karen; Hell, Johannes W

    2016-01-01

    Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction. PMID:27303637

  18. Downscaling the Analysis of Complex Transmembrane Signaling Cascades to Closed Attoliter Volumes

    PubMed Central

    Grasso, Luigino; Wyss, Romain; Piguet, Joachim; Werner, Michael; Hassaïne, Ghérici; Hovius, Ruud; Vogel, Horst

    2013-01-01

    Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format. PMID:23940670

  19. Non-ionotropic signaling by the NMDA receptor: controversy and opportunity.

    PubMed

    Gray, John A; Zito, Karen; Hell, Johannes W

    2016-01-01

    Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction.

  20. Non-ionotropic signaling by the NMDA receptor: controversy and opportunity

    PubMed Central

    Gray, John A.; Zito, Karen; Hell, Johannes W.

    2016-01-01

    Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction. PMID:27303637

  1. P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

    PubMed

    Adamczyk, Magdalena; Griffiths, Rhiannon; Dewitt, Sharon; Knäuper, Vera; Aeschlimann, Daniel

    2015-12-15

    Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell-matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

  2. Smooth muscle length-dependent PI(4,5)P2 synthesis and paxillin tyrosine phosphorylation.

    PubMed

    Sul, D; Baron, C B; Broome, R; Coburn, R F

    2001-07-01

    We studied effects of increasing the length of porcine trachealis muscle on 5.5 microM carbachol (CCh)-evoked phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] synthesis and other parameters of phosphatidylinositol (PI) turnover. PI(4,5)P2 resynthesis rates in muscle held at 1.0 optimal length (L(o)), measured over the first 6 min of CCh stimulation, were 140 +/- 12 and 227 +/- 14% of values found in muscle held at 0.5 L(o) and in free-floating muscle, respectively. Time-dependent changes in cellular masses of PI(4,5)P2, PI, and phosphatidic acid, and PI resynthesis rates, were also altered by the muscle length at which contraction occurred. In free-floating muscle, CCh did not evoke increases in tyrosine-phosphorylated paxillin (PTyr-paxillin), an index of beta1-integrin signaling; however, there were progressive increases in PTyr-paxillin in muscle held at 0.5 and 1.0 L(o) during contraction, which correlated with increases in PI(4,5)P2 synthesis rates. These data indicate that PI(4,5)P2 synthesis rates and other parameters of CCh-stimulated inositol phospholipid turnover are muscle length-dependent and provide evidence that supports the hypothesis that length-dependent beta1-integrin signals may exert control on CCh-activated PI(4,5)P2 synthesis.

  3. P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2

    PubMed Central

    Adamczyk, Magdalena; Griffiths, Rhiannon; Dewitt, Sharon; Knäuper, Vera; Aeschlimann, Daniel

    2015-01-01

    ABSTRACT Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell–matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca2+ signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions. PMID:26542019

  4. Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

    SciTech Connect

    Slivka, S.R.; Insel, P.A.

    1987-03-25

    alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.

  5. Behavior-linked FoxP2 regulation enables zebra finch vocal learning.

    PubMed

    Heston, Jonathan B; White, Stephanie A

    2015-02-18

    Mutations in the FOXP2 transcription factor cause an inherited speech and language disorder, but how FoxP2 contributes to learning of these vocal communication signals remains unclear. FoxP2 is enriched in corticostriatal circuits of both human and songbird brains. Experimental knockdown of this enrichment in song control neurons of the zebra finch basal ganglia impairs tutor song imitation, indicating that adequate FoxP2 levels are necessary for normal vocal learning. In unmanipulated birds, vocal practice acutely downregulates FoxP2, leading to increased vocal variability and dynamic regulation of FoxP2 target genes. To determine whether this behavioral regulation is important for song learning, here, we used viral-driven overexpression of FoxP2 to counteract its downregulation. This manipulation disrupted the acute effects of song practice on vocal variability and caused inaccurate song imitation. Together, these findings indicate that dynamic behavior-linked regulation of FoxP2, rather than absolute levels, is critical for vocal learning.

  6. P2X7 Mediates ATP-Driven Invasiveness in Prostate Cancer Cells

    PubMed Central

    Qiu, Ying; Li, Wei-hua; Zhang, Hong-quan; Liu, Yan; Tian, Xin-Xia; Fang, Wei-Gang

    2014-01-01

    The ATP-gated P2X7 has been shown to play an important role in invasiveness and metastasis of some tumors. However, the possible links and underlying mechanisms between P2X7 and prostate cancer have not been elucidated. Here, we demonstrated that P2X7 was highly expressed in some prostate cancer cells. Down-regulation of P2X7 by siRNA significantly attenuated ATP- or BzATP-driven migration and invasion of prostate cancer cells in vitro, and inhibited tumor invasiveness and metastases in nude mice. In addition, silencing of P2X7 remarkably attenuated ATP- or BzATP- driven expression changes of EMT/invasion-related genes Snail, E-cadherin, Claudin-1, IL-8 and MMP-3, and weakened the phosphorylation of PI3K/AKT and ERK1/2 in vitro. Similar effects were observed in nude mice. These data indicate that P2X7 stimulates cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes, as well as PI3K/AKT and ERK1/2 signaling pathways. P2X7 could be a promising therapeutic target for prostate cancer. PMID:25486274

  7. Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage

    PubMed Central

    Cisneros-Mejorado, Abraham; Gottlieb, Miroslav; Cavaliere, Fabio; Magnus, Tim; Koch-Nolte, Friederich; Scemes, Eliana; Pérez-Samartín, Alberto; Matute, Carlos

    2015-01-01

    The role of P2X7 receptors and pannexin-1 channels in ischemic damage remains controversial. Here, we analyzed their contribution to postanoxic depolarization after ischemia in cultured neurons and in brain slices. We observed that pharmacological blockade of P2X7 receptors or pannexin-1 channels delayed the onset of postanoxic currents and reduced their slope, and that simultaneous inhibition did not further enhance the effects of blocking either one. These results were confirmed in acute cortical slices from P2X7 and pannexin-1 knockout mice. Oxygen-glucose deprivation in cortical organotypic cultures caused neuronal death that was reduced with P2X7 and pannexin-1 blockers as well as in organotypic cultures derived from mice lacking P2X7 and pannexin 1. Subsequently, we used transient middle cerebral artery occlusion to monitor the neuroprotective effect of those drugs in vivo. We found that P2X7 and pannexin-1 antagonists, and their ablation in knockout mice, substantially attenuated the motor symptoms and reduced the infarct volume to ~50% of that in vehicle-treated or wild-type animals. These results show that P2X7 receptors and pannexin-1 channels are major mediators of postanoxic depolarization in neurons and of brain damage after ischemia, and that they operate in the same deleterious signaling cascade leading to neuronal and tissue demise. PMID:25605289

  8. The Critical Role Of VP1 In Forming The Necessary Cavities For Receptor-mediated Entry Of FMDV To The Host Cell.

    PubMed

    Ashkani, Jahanshah; Rees, D J G

    2016-01-01

    The antigenic inconsistency of the foot-and-mouth disease virus (FMDV) is very broad, such that a vaccine made from one isolate will not offer protection against infection with other isolates from the same serotype. Viral particles (VPs) or surface exposed capsid proteins, VP1-VP3, of FMDV determine both the antigenicity of the virus and its receptor-mediated entry into the host cell. Therefore, modifications of these structural proteins may alter the properties of the virus. Here we show putative cavities on the FMDV-SAT1 (FMDV Southern African Territories1) capsid as possible binding sites for the receptor-mediated viral entry into the host cell. We identified three possible cavities on the FMDV capsid surface, from which the largest one (C2) is shaped in the contact regions of VP1-VP3. Our results demonstrate the significance of VP1, in the formation of FMDV-SAT1 surface cavities, which is the main component in all the identified cavities. Our findings can have profound implications in the protein engineering of FMDV in the contact region of VP1-VP3 found to be embedded in several cavities. Such information is of great significance in the context of vaccine design, as it provides the ground for future improvement of synthetic vaccines to control FMD caused by FMDV-SAT1 serotypes. PMID:27249937

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