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Sample records for p2x7 receptors mediate

  1. 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

  2. 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

  3. The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System*

    PubMed Central

    García-Huerta, Paula; Díaz-Hernandez, Miguel; Delicado, Esmerilda G.; Pimentel-Santillana, María; Miras-Portugal, Mª Teresa; Gómez-Villafuertes, Rosa

    2012-01-01

    P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites. PMID:23139414

  4. 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.

  5. Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages

    NASA Astrophysics Data System (ADS)

    Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Kmit, Arthur; Romao, Ana M.; Jantarajit, Walailak; Schreiber, Rainer; Kunzelmann, Karl

    2015-02-01

    Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca2+ dependent phospholipid scramblase and Ca2+-activated Cl- channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.

  6. Homodimeric anoctamin-1, but not homodimeric anoctamin-6, is activated by calcium increases mediated by the P2Y1 and P2X7 receptors.

    PubMed

    Stolz, Michaela; Klapperstück, Manuela; Kendzierski, Thomas; Detro-Dassen, Silvia; Panning, Anna; Schmalzing, Günther; Markwardt, Fritz

    2015-10-01

    The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum.

  7. Homodimeric anoctamin-1, but not homodimeric anoctamin-6, is activated by calcium increases mediated by the P2Y1 and P2X7 receptors.

    PubMed

    Stolz, Michaela; Klapperstück, Manuela; Kendzierski, Thomas; Detro-Dassen, Silvia; Panning, Anna; Schmalzing, Günther; Markwardt, Fritz

    2015-10-01

    The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum. PMID:25592660

  8. 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

  9. 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

  10. Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors

    PubMed Central

    Nörenberg, W; Sobottka, H; Hempel, C; Plötz, T; Fischer, W; Schmalzing, G; Schaefer, M

    2012-01-01

    BACKGROUND AND PURPOSE In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca2+]i influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG+ permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues. PMID:22506590

  11. 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

  12. 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

  13. 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

  14. P2X7 Receptor Function in Bone-Related Cancer

    PubMed Central

    Adinolfi, Elena; Amoroso, Francesca; Giuliani, Anna Lisa

    2012-01-01

    Modulation of tumor microenvironment by different mediators is central in determining neoplastic formation and progression. Among these molecules extracellular ATP is emerging as a good candidate in promoting cell growth, neovascularization, tumor-host interactions, and metastatization. This paper summarizes recent findings on expression and function of P2X7 receptor for extracellular ATP in primary and metastatic bone cancers. Search of mRNA expression microchip databases and literature analysis demonstrate a high expression of P2X7 in primary bone tumors as well as in other malignancies such as multiple myeloma, neuroblastoma, breast, and prostate cancer. Evidence that P2X7 triggers NFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting either primary tumor development or osteoblastic lesions is also reported. Moreover, P2X7 receptor is involved in osteoclast differentiation, RANKL expression, matrix metalloproteases and cathepsin secretion thus promoting bone resorption and osteolytic lesions. Taken together these data point to a pivotal role for the P2X7 receptor in bone cancer biology. PMID:22970409

  15. Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia

    PubMed Central

    Nörenberg, W; Schunk, J; Fischer, W; Sobottka, H; Riedel, T; Oliveira, JF; Franke, H; Illes, P

    2010-01-01

    Background and purpose: P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. Experimental approach: We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial–neuronal co-cultures prepared from the cerebral cortex of rats. Key results: All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [α,β-methylene ATP and 2′-3′-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5′-O-(2-thiodiphosphate), uridine 5′-diphosphate, uridine 5′-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. Conclusions and implications: Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury. PMID:20649592

  16. 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.

  17. 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

  18. Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists.

    PubMed

    Caseley, Emily A; Muench, Stephen P; Fishwick, Colin W; Jiang, Lin-Hua

    2016-09-15

    The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3μM, 4.8±0.8μM and 3.2±0.2μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3μM and 1.2±0.1μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9μM, 1.0±0.1μM and 0.8±0.2μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation. PMID:27481062

  19. Trophic activity of human P2X7 receptor isoforms A and B in osteosarcoma.

    PubMed

    Giuliani, Anna Lisa; Colognesi, Davide; Ricco, Tiziana; Roncato, Carlotta; Capece, Marina; Amoroso, Francesca; Wang, Qi Guang; De Marchi, Elena; Gartland, Allison; Di Virgilio, Francesco; Adinolfi, Elena

    2014-01-01

    The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca(2+) mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently

  20. P2X7 receptor in epilepsy; role in pathophysiology and potential targeting for seizure control

    PubMed Central

    Engel, Tobias; Jimenez-Pacheco, Alba; Miras-Portugal, Maria Teresa; Diaz-Hernandez, Miguel; Henshall, David C

    2012-01-01

    The P2X7 receptor is an ATP-gated non-selective cation-permeable ionotropic receptor selectively expressed in neurons and glia in the brain. Activation of the P2X7 receptor has been found to modulate neuronal excitability in the hippocampus and it has also been linked to microglia activation and neuroinflammatory responses. Accordingly, interest developed on the P2X7 receptor in disorders of the nervous system, including epilepsy. Studies show that expression of the P2X7 receptor is elevated in damaged regions of the brain after prolonged seizures (status epilepticus) in both neurons and glia. P2X7 receptor expression is also increased in the hippocampus in experimental epilepsy. Recent data show that mice lacking the P2X7 receptor display altered susceptibility to status epilepticus and that drugs targeting the P2X7 receptor have potent anticonvulsant effects. Together, this suggests that P2X7 receptor ligands may be useful adjunctive treatments for refractory status epilepticus or perhaps pharmacoresistant epilepsy. This review summarizes the evidence of P2X7 receptor involvement in the pathophysiology of epilepsy and the potential of drugs targeting this receptor for seizure control. PMID:23320131

  1. 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

  2. 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

  3. 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

  4. P2X7 receptor as predictor gene for glioma radiosensitivity and median survival.

    PubMed

    Gehring, Marina P; Kipper, Franciele; Nicoletti, Natália F; Sperotto, Nathalia D; Zanin, Rafael; Tamajusuku, Alessandra S K; Flores, Debora G; Meurer, Luise; Roesler, Rafael; Filho, Aroldo B; Lenz, Guido; Campos, Maria M; Morrone, Fernanda B

    2015-11-01

    Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival. PMID:26358881

  5. Accelerated tumor progression in mice lacking the ATP receptor P2X7.

    PubMed

    Adinolfi, Elena; Capece, Marina; Franceschini, Alessia; Falzoni, Simonetta; Giuliani, Anna L; Rotondo, Alessandra; Sarti, Alba C; Bonora, Massimo; Syberg, Susanne; Corigliano, Domenica; Pinton, Paolo; Jorgensen, Niklas R; Abelli, Luigi; Emionite, Laura; Raffaghello, Lizzia; Pistoia, Vito; Di Virgilio, Francesco

    2015-02-15

    The ATP receptor P2X7 (P2X7R or P2RX7) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study, we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with postmortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intratumoral IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an antitumor immune response, and to restrict tumor growth and metastatic diffusion. PMID:25542861

  6. P2X7 R-mediated Ca(2+) -independent d-serine release via pannexin-1 of the P2X7 R-pannexin-1 complex in astrocytes.

    PubMed

    Pan, Han-Chi; Chou, Yun-Chia; Sun, Synthia H

    2015-05-01

    D-serine is a coagonist of N-methyl-d-aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity-dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d-serine from astrocytes and discovered a novel Ca(2+) -independent release mechanism mediated by P2X7 receptors (P2X7 R). Using [(3) H] d-serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X7 R agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), stimulated [(3) H]D-serine release and that were abolished by P2X7 R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca(2+) . The P2X7 R-mediated d-serine release was inhibited by pannexin-1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and (10) Panx-1 peptide, and shRNAs, and stimulation of P2X7 R induced P2X7 R-pannexin-1 complex formation. Simply incubating astrocytes in Ca(2+) /Mg(2+) -free buffer also induced the complex formation, and that enhanced basal d-serine release through pannexin-1. The P2X7 R-mediated d-serine release assayed in Ca(2+) /Mg(2+) -free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca(2+) -dependent PKC inhibitor, Gö6976, inhibited the P2X7 R-mediated d-serine release. Thus, we conclude that in astrocytes, P2X7 R-pannexin-1 complex formation is crucial for P2X7 R-mediated d-serine release through pannexin-1 hemichannel. The release is Ca(2+) -independent and regulates by a Ca(2+) -independent PKC. The activated P2X7 R per se is also functioned as a permeation channel to release d-serine in part. This P2X7 R-mediated d-serine release represents an important mechanism for activity-dependent neuron-glia interaction.

  7. 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

  8. 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.

  9. 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.

  10. Potent Suppressive Effects of 1-Piperidinylimidazole Based Novel P2X7 Receptor Antagonists on Cancer Cell Migration and Invasion.

    PubMed

    Park, Jin-Hee; Williams, Darren R; Lee, Ji-Hyung; Lee, So-Deok; Lee, Je-Heon; Ko, Hyojin; Lee, Ga-Eun; Kim, Sujin; Lee, Jeong-Min; Abdelrahman, Aliaa; Müller, Christa E; Jung, Da-Woon; Kim, Yong-Chul

    2016-08-25

    The P2X7 receptor (P2X7R) has been reported as a key mediator in inflammatory processes and cancer invasion/metastasis. In this study, we report the discovery of novel P2X7R antagonists and their functional activities as potential antimetastatic agents. Modifications of the hydantoin core-skeleton and the side chain substituents of the P2X7R antagonist 7 were performed. The structure-activity relationships (SAR) and optimization demonstrated the importance of the sulfonyl group at the R1 position and the substituted position and overall size of R2 for P2X7R antagonism. The optimized novel analogues displayed potent P2X7 receptor antagonism (IC50 = 0.11-112 nM) along with significant suppressive effects on IL-1β release (IC50 = 0.32-210 nM). Moreover, representative antagonists (12g, 13k, and 17d) with imidazole and uracil core skeletons significantly inhibited the invasion of MDA-MB-231 triple negative breast cancer cells and cancer cell migration in a zebrafish xenograft model, suggesting the potential therapeutic application of these novel P2X7 antagonists to block metastatic cancer. PMID:27427902

  11. Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse.

    PubMed

    Vázquez-Cuevas, F G; Cruz-Rico, A; Garay, E; García-Carrancá, A; Pérez-Montiel, D; Juárez, B; Arellano, R O

    2013-01-01

    Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

  12. Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

    PubMed

    Giannuzzo, Andrea; Saccomano, Mara; Napp, Joanna; Ellegaard, Maria; Alves, Frauke; Novak, Ivana

    2016-12-01

    The ATP-gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu-1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu-1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7(-/-) animals. PancTu-1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120-treated mice showed reduced bioluminescence compared to saline-treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120-treated tumours. PMID:27513892

  13. Effects of antidepressants on P2X7 receptors.

    PubMed

    Wang, Wei; Xiang, Zheng-Hua; Jiang, Chun-Lei; Liu, Wei-Zhi; Shang, Zhi-Lei

    2016-08-30

    Antidepressants including paroxetine, fluoxetine and desipramine are commonly used for treating depression. P2×7 receptors are member of the P2X family. Recent studies indicate that these receptors may constitute a novel potential target for the treatment of depression. In the present study, we examined the action of these antidepressants on cloned rat P2×7 receptors that were stably expressed in human embryonic kidney (HEK) 293 cells by using the whole-cell patch-clamp technique, and found that paroxetine at a dose of 10µM could significantly reduce the inward currents evoked by the P2×7 receptors agonist BzATP by pre-incubation for 6-12 but not by acute application (10µM) or pre-incubation for 2-6h at a dose of 1µM, 3µM or 10µM paroxetine. Neither fluoxetine nor desipramine had significant effects on currents evoked by BzATP either applied acutely or by pre-incubation at various concentrations. These results suggest that the sensitivity of rat P2×7 receptors to antidepressants is different, which may represent an unknown mechanism by which these drugs exert their therapeutic effects and side effects. PMID:27318632

  14. Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: A pain mechanism mediated by Pannexin 1.

    PubMed

    Di Cesare Mannelli, Lorenzo; Marcoli, Manuela; Micheli, Laura; Zanardelli, Matteo; Maura, Guido; Ghelardini, Carla; Cervetto, Chiara

    2015-10-01

    Anticancer therapy based on the repeated administration of oxaliplatin is limited by the development of a neuropathic syndrome difficult to treat. Oxaliplatin neurotoxicity is based on complex nervous mechanisms, the comprehension of the role of single neurotransmitters and the knowledge of the signal flow among cells is matter of importance to improve therapeutic chances. In a rat model of oxaliplatin-induced neuropathy, we report increased P2X7-evoked glutamate release from cerebrocortical synaptosomes. The release was abolished by the P2X7 receptor (P2X7R) antagonists Brilliant-Blue-G (BBG) and A-438079, and significantly reduced by Carbenoxolone and the Pannexin 1 (Panx1) selective inhibitors Erioglaucine and (10)Panx suggesting the recruitment of Panx1. Aimed to evaluate the significance of P2X7R-Panx1 system activation in pain generated by oxaliplatin, pharmacological modulators were spinally infused by intrathecal catheter in oxaliplatin-treated animals. BBG, Erioglaucine and (10)Panx reverted oxaliplatin-dependent pain. Finally, the influence of the P2X7R-Panx1 system blockade on oxaliplatin anticancer activity was evaluated on the human colon cancer cell line HT-29. Prevention of HT-29 apoptosis and mortality was dependent by kind and concentration of P2X7R antagonists. On the contrary, the inhibition of Panx1 did not alter oxaliplatin lethality in tumor cells. It is concluded that glutamate release dependent on P2X7R is increased in cerebrocortical nerve terminals from oxaliplatin-treated rats; the increase is mediated by functional recruitment of Panx1; P2X7R antagonists and Panx1 inhibitors revert oxaliplatin-induced neuropathic pain; Panx1 inhibitors do not alter the oxaliplatin-induced mortality of cancer cells HT-29. The inhibition of Panx1 channel is suggested as a new and safe pharmacological target.

  15. Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration.

    PubMed

    Alvarez, Alvaro; Lagos-Cabré, Raúl; Kong, Milene; Cárdenas, Areli; Burgos-Bravo, Francesca; Schneider, Pascal; Quest, Andrew F G; Leyton, Lisette

    2016-09-01

    Our previous reports indicate that ligand-induced αVβ3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca(2+) to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVβ3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVβ3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca(2+), hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVβ3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca(2+) entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

  16. Activation of P2X7 receptors in the midbrain periaqueductal gray of rats facilitates morphine tolerance.

    PubMed

    Xiao, Zhi; Li, You-Yan; Sun, Meng-Jie

    2015-08-01

    Opiates such as morphine exhibit analgesic effect in various pain models, but repeated and chronic morphine administration may develop resistance to antinociception. The purinergic signaling system is involved in the mechanisms of pain modulation and morphine tolerance. This study aimed to determine whether the P2X7 receptor in the ventrolateral midbrain periaqueductal gray (vlPAG) is involved in morphine tolerance. Development of tolerance to the antinociceptive effect of morphine was induced in normal adult male Sprague-Dawley (SD) rats through subcutaneous injection of morphine (10mg/kg). The analgesic effect of morphine (5mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds (MWTs) in rats with an electronic von Frey anesthesiometer. The expression levels and distribution of the P2X7 receptor in the vlPAG was evaluated through Western blot analysis and immunohistochemistry. The acute effects of intra-vlPAG injection of the selective P2X7 receptor agonist Bz-ATP, the selective P2X7 receptor antagonist A-740003, or antisense oligodeoxynucleotide (AS ODN) targeting the P2X7 receptor on morphine-treated rats were also observed. Results demonstrated that repeated morphine administration decreased the mechanical pain thresholds. By contrast, the expression of the P2X7 receptor protein was up-regulated in the vlPAG in morphine tolerant rats. The percent changes in MWT were markedly but only transiently attenuated by intra-vlPAG injection of Bz-ATP (9nmol/0.3μL) but elevated by A-740003 at doses of 10 and 100nmol/0.3μL. AS ODN (15nmol/0.3μL) against the P2X7 receptor reduced the development of chronic morphine tolerance in rats. These results suggest that the development of antinociceptive tolerance to morphine is partially mediated by activating the vlPAG P2X7 receptors. The present data also suggest that the P2X7 receptors may be a therapeutic target for improving the analgesic effect of morphine in treatments of pain when morphine tolerance

  17. Regulation of the P2X7 receptor permeability to large molecules by extracellular Cl- and Na+.

    PubMed

    Li, Qin; Luo, Xiang; Muallem, Shmuel

    2005-07-22

    Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na

  18. Critical Evaluation of P2X7 Receptor Antagonists in Selected Seizure Models

    PubMed Central

    Fischer, Wolfgang; Franke, Heike; Krügel, Ute; Müller, Heiko; Dinkel, Klaus; Lord, Brian; Letavic, Michael A.; Henshall, David C.; Engel, Tobias

    2016-01-01

    The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders. PMID:27281030

  19. N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1β Release from Murine Macrophages

    PubMed Central

    2016-01-01

    Extracellular adenosine 5′-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1β release from murine J774 macrophages. ATP-induced IL-1β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-{4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment. PMID:27524862

  20. 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.

  1. Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567

    PubMed Central

    Bhattacharya, Anindya; Wang, Qi; Ao, Hong; Shoblock, James R; Lord, Brian; Aluisio, Leah; Fraser, Ian; Nepomuceno, Diane; Neff, Robert A; Welty, Natalie; Lovenberg, Timothy W; Bonaventure, Pascal; Wickenden, Alan D; Letavic, Michael A

    2013-01-01

    BACKGROUND AND PURPOSE An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist. EXPERIMENTAL APPROACH We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain. KEY RESULTS JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL−1 (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg−1) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test. Conclusion and Implications JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology. PMID:23889535

  2. [Effect of P2X7 receptor knock-out on bone cancer pain in mice].

    PubMed

    Zhao, Xin; Liu, Hui-Zhu; Zhang, Yu-Qiu

    2016-06-25

    Cancer pain is one of the most common symptoms in patients with late stage cancer. Lung, breast and prostate carcinoma are the most common causes of pain from osseous metastasis. P2X7 receptor (P2X7R) is one of the subtypes of ATP-gated purinergic ion channel family, predominately distributed in microglia in the spinal cord. Activation of P2X7Rs in the spinal dorsal horn has been associated with release of proinflammatory cytokines from glial cells, causing increased neuronal excitability and exaggerated nociception. Mounting evidence implies a critical role of P2X7R in inflammatory and neuropathic pain. However, whether P2X7R is involved in cancer pain remains controversial. Here we established a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6J wild-type mice (C57 WT mice) and P2X7R knockout mice (P2rx7(-/-) mice) to explore the role of P2X7R in bone cancer pain. Following intrafemur carcinoma inoculation, robust mechanical allodynia and thermal hyperalgesia in C57 WT mice were developed on day 7 and 14, respectively, and persisted for at least 28 days in the ipsilateral hindpaw of the affected limb. CatWalk gait analysis showed significant decreases in the print area and stand phase, and a significant increase in swing phase in the ipsilateral hindpaw on day 21 and 28 after carcinoma cells inoculation. Histopathological sections (hematoxylin and eosin stain) showed that the bone marrow of the affected femur was largely replaced by invading tumor cells, and the femur displayed medullary bone loss and bone destruction on day 28 after inoculation. Unexpectedly, no significant changes in bone cancer-induced hypersensitivity of pain behaviors were found in P2rx7(-/-) mice, and the changes of pain-related values in CatWalk gait analysis even occurred earlier in P2rx7(-/-) mice, as compared with C57 WT mice. Together with our previous study in rats that blockade of P2X7R significantly alleviated bone cancer

  3. A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection

    PubMed Central

    Ramos-Junior, E.S.; Morandini, A.C.; Almeida-da-Silva, C.L.C.; Franco, E.J.; Potempa, J.; Nguyen, K.A.; Oliveira, A.C.; Zamboni, D.S.; Ojcius, D.M.; Scharfstein, J.

    2015-01-01

    Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1β and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1β secretion by means of its fimbriae in a purinergic P2X7 receptor–dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1β processing and secretion by P. gingivalis–infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1β secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1β from P. gingivalis–infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1β to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1β secretion but also for intracellular pro-IL-1β processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7-/- mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis. PMID:26152185

  4. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  5. Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

    PubMed Central

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  6. 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

  7. The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor.

    PubMed

    Hempel, Christoph; Nörenberg, Wolfgang; Sobottka, Helga; Urban, Nicole; Nicke, Annette; Fischer, Wolfgang; Schaefer, Michael

    2013-12-01

    P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

  8. Involvement of RVM-expressed P2X7 receptor in bone cancer pain: mechanism of descending facilitation.

    PubMed

    Huang, Zhang Xiang; Lu, Zhi Jie; Ma, Wei Qing; Wu, Fei Xiang; Zhang, Yu Qiu; Yu, Wei-Feng; Zhao, Zhi Qi

    2014-04-01

    Patients with bone cancer commonly experience bone pain that is severe, intolerable, and difficult to manage. The rostral ventromedial medulla (RVM) plays an important role in the development of chronic pain via descending facilitation of spinal nociception. The compelling evidence shows that glial P2X7 receptor (P2X7R) is involved in the induction and maintenance of chronic pain syndromes. The present study explored the mechanism of glial activation and P2X7R expression underlying the induction of bone cancer pain. The results demonstrated that microglia and astrocytes in the RVM were markedly activated in bone cancer rats, and the expression of P2X7R was significantly upregulated. Injection of Brilliant Blue G (BBG), an inhibitor of P2X7R, into the RVM significantly alleviated pain behaviors of cancer rats, which was supported by intra-RVM injection of RNA interference targeting the P2X7R in the RVM. It is suggested that activation of microglia-expressed P2X7R in the RVM contributes to bone cancer pain. Given that 5-HT in the RVM is involved in modulating spinal nociception, changes in 5-HT and Fos expression were addressed in the spinal cord. Inhibition of P2X7R by BBG or small-interference RNA targeting P2X7 in the RVM markedly reduced 5-HT level and Fos expression in the spinal cord. The data clearly suggest that the activation of microglial P2X7R in the RVM contributes to the development of bone cancer pain via upregulation of spinal 5HT levels by the descending pain facilitatory system.

  9. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  10. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+) T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+) T cell immunity.

  11. P2X7 receptor antagonism improves renal blood flow and oxygenation in angiotensin-II infused rats

    PubMed Central

    Menzies, Robert I.; Howarth, Amelia R.; Unwin, Robert J.; Tam, Frederick W.K.; Mullins, John J.; Bailey, Matthew A.

    2015-01-01

    Chronic activation of the renin angiotensin system promotes hypertension, renal microvascular dysfunction, tissue hypoxia and inflammation. We found previously that the injurious response to excess angiotensin II (ANGII) is greater in F344 rats, whereas Lewis rats are renoprotected, despite similar hypertension. We further identified p2rx7, encoding the P2X7 receptor (P2X7R), as a candidate gene for differential susceptibility and here we have tested the hypothesis that activation of P2X7R promotes vascular dysfunction under high ANGII tone. 14-day infusion of ANGII at 30ng/min into F344 rats increased blood pressure by ~15mmHg without inducing fibrosis or albuminuria. In vivo pressure natriuresis was suppressed, medullary perfusion reduced by ~50% and the cortico-medullary oxygenation gradient disrupted. Selective P2X7R antagonism restored pressure natriuresis, promoting a significant leftward shift in the intercept and increasing the slope. Sodium excretion was increased 6 fold and blood pressure normalized. The specific P2X7R antagonist AZ11657312 increased renal medullary perfusion, but only in ANGII-treated rats. Tissue oxygenation was improved by P2X7R blockade, particularly in poorly oxygenated regions of the kidney. Activation of P2X7R induces microvascular dysfunction and regional hypoxia when ANGII is elevated. These pro-inflammatory effects may contribute to progression of renal injury induced by chronic ANGII. PMID:26108066

  12. The C-Terminus of Human Nucleotide Receptor P2X7 Is Critical for Receptor Oligomerization and N-Linked Glycosylation

    PubMed Central

    Wickert, Lisa E.; Blanchette, Joshua B.; Waldschmidt, Noelle V.; Bertics, Paul J.; Denu, John M.; Denlinger, Loren C.; Lenertz, Lisa Y.

    2013-01-01

    Background The P2X7 receptor binds extracellular ATP to mediate numerous inflammatory responses and is considered a potential biomarker and therapeutic target for diverse inflammatory and neurological diseases. P2X7 contains many single nucleotide polymorphisms, including several mutations located within its intracellular C-terminal trafficking domain. Mutations within the trafficking domain result in attenuated receptor activity and cell surface presentation, but the mechanisms by which amino acid changes within this region promote altered P2X7 function have not been elucidated. Methods and Results We analyzed the amino acid sequence of P2X7 for any potential trafficking signals and found that P2X7 contains putative Arg-X-Arg ER retention sequences. Alanine substitutions near or within these sequences were constructed, and we determined that single mutation of R574 and R578 but not R576 or K579 attenuates P2X7-stimulated activation of ERK1/2 and induction of the transcription factors FosB and ΔFosB. We found that mutation of R578 within the trafficking domain to the naturally occurring Gln substitution disrupts P2X7 localization at the plasma membrane and results in R578Q displaying a higher apparent molecular weight in comparison to wild-type receptor. We used the glycosidase endoglycosidase H to determine that this difference in mass is due in part to the R578Q mutant possessing a larger mass of oligosaccharides, indicative of improper N-linked glycosylation addition and/or trimming. Chemical cross-linking experiments were also performed and suggest that the R578Q variant also does not form trimers as well as wild-type receptor, a function required for its full activity. Conclusions These data demonstrate the distal C-terminus of P2X7 is important for oligomerization and post-translational modification of the receptor, providing a mechanism by which mutations in the trafficking domain disrupt P2X7 activity and localization at the plasma membrane. PMID:23691096

  13. 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

  14. 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.

  15. Shockwaves induce osteogenic differentiation of human mesenchymal stem cells through ATP release and activation of P2X7 receptors.

    PubMed

    Sun, Dahui; Junger, Wolfgang G; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2013-06-01

    Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (≈ 7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.

  16. Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors

    PubMed Central

    Sun, Dahui; Junger, Wolfgang G.; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2014-01-01

    Shockwave fractures treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5’-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK sig naling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (~7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation. PMID:23404811

  17. Caspase-11 requires the pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock

    PubMed Central

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2016-01-01

    SUMMARY The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel and ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by LPS transfection or treatment with cholera toxin B and LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11−/−, Panx1−/− or P2x7−/− mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. PMID:26572062

  18. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock.

    PubMed

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2015-11-17

    The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. PMID:26572062

  19. P2X7 receptor and caspase 1 activation are central to airway inflammation observed after exposure to tobacco smoke.

    PubMed

    Eltom, Suffwan; Stevenson, Christopher S; Rastrick, Joseph; Dale, Nicole; Raemdonck, Kristof; Wong, Sissie; Catley, Matthew C; Belvisi, Maria G; Birrell, Mark A

    2011-01-01

    Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently there is no effective medication to stop the relentless progression of this disease. It has recently been shown that an activator of the P2X7/inflammasome pathway, ATP, and the resultant products (IL-1β/IL-18) are increased in COPD patients. The aim of this study was to determine whether activation of the P2X7/caspase 1 pathway has a functional role in CS-induced airway inflammation. Mice were exposed to CS twice a day to induce COPD-like inflammation and the role of the P2X7 receptor was investigated. We have demonstrated that CS-induced neutrophilia in a pre-clinical model is temporally associated with markers of inflammasome activation, (increased caspase 1 activity and release of IL-1β/IL-18) in the lungs. A selective P2X7 receptor antagonist and mice genetically modified so that the P2X7 receptors were non-functional attenuated caspase 1 activation, IL-1β release and airway neutrophilia. Furthermore, we demonstrated that the role of this pathway was not restricted to early stages of disease development by showing increased caspase 1 activation in lungs from a more chronic exposure to CS and from patients with COPD. This translational data suggests the P2X7/Inflammasome pathway plays an ongoing role in disease pathogenesis. These results advocate the critical role of the P2X7/caspase 1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD. PMID:21915284

  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. Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons.

    PubMed

    Ohishi, A; Keno, Y; Marumiya, A; Sudo, Y; Uda, Y; Matsuda, K; Morita, Y; Furuta, T; Nishida, K; Nagasawa, K

    2016-04-01

    Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

  2. An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor.

    PubMed

    Gu, Ben J; Sluyter, Ronald; Skarratt, Kristen K; Shemon, Anne N; Dao-Ung, Lan-Phuong; Fuller, Stephen J; Barden, Julian A; Clarke, Alison L; Petrou, Steven; Wiley, James S

    2004-07-23

    The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7). PMID:15123679

  3. ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

    PubMed Central

    Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2011-01-01

    Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

  4. 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

  5. 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

    PubMed

    Rubio-Araiz, Ana; Perez-Hernandez, Mercedes; Urrutia, Andrés; Porcu, Francesca; Borcel, Erika; Gutierrez-Lopez, Maria Dolores; O'Shea, Esther; Colado, Maria Isabel

    2014-08-01

    The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1β levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

  6. P2X7 receptor predicts postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma

    PubMed Central

    Liu, Zheng; Liu, Yidong; Xu, Le; An, Huimin; Chang, Yuan; Yang, Yuanfeng; Zhang, Weijuan; Xu, Jiejie

    2015-01-01

    The P2X7 receptor, an ATP-gated plasma membrane ion channel, is involved in inflammation, apoptosis and cell proliferation, and thereby plays a crucial role during oncogenic transformation in various malignancies. This study aims to evaluate the impact of P2X7 receptor expression on postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma (ccRCC). A total of 273 patients with ccRCC undergoing nephrectomy at a single institution were retrospectively enrolled in this study, among which 86 patients died of this disease and six patients died of other causes. Clinicopathologic features and cancer-specific survival (CSS) were recorded. P2X7 expression was assessed by immunohistochemistry in clinical specimens. Kaplan–Meier method with log rank test was performed to compare survival curves. Cox regression models were used to evaluate the prognostic values of variables on CSS. Concordance index was calculated to assess prognostic accuracy of prognostic models. Median follow-up period was 90 months (range, 11–120 months). Intratumoral P2X7 expression was significantly lower than peritumoral tissues (P < 0.001). Moreover, high intratumoral P2X7 expression, which was significantly associated with shorten CSS (P < 0.001), high TNM stage (P = 0.038), Fuhrman grade (P = 0.035), SSIGN (stage, size, grade, and necrosis) score (P = 0.021) and University of California Integrated Staging System (UISS) score (P = 0.007), was indicated to be an independent prognostic factor for CSS (hazard ratio [HR], 1.693; P = 0.034). The prognostic accuracy of TNM stage, UISS and SSIGN scoring models was improved when intratumoral P2X7 expression was added. Intratumoral P2X7 expression is a potential independent adverse prognostic indicator for postoperative CSS of patients with ccRCC. PMID:26179886

  7. FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

    PubMed

    Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

    2013-02-01

    Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells.

  8. FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

    PubMed

    Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

    2013-02-01

    Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. PMID:22806078

  9. Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors.

    PubMed

    Csölle, Cecilia; Baranyi, Mária; Zsilla, Gabriella; Kittel, Agnes; Gölöncsér, Flóra; Illes, Peter; Papp, Edit; Vizi, E Sylvester; Sperlágh, Beáta

    2013-01-01

    Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7-/-) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [(3)H]glutamate in P2rx7+/+ but not P2rx7-/- mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7-/- mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7-/- mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7-/- mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7-/- mice, which accompanied the increased uptake of [(3)H]5-HT and an elevated number of [(3)H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7-/- mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7-/- mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.

  10. A Reassessment of P2X7 Receptor Inhibition as a Neuroprotective Strategy in Rat Models of Contusion Injury

    PubMed Central

    Marcillo, Alexander; Frydel, Beata; Bramlett, Helen M.; Dietrich, W. Dalton

    2011-01-01

    These experiments were completed as part of an NIH “Facilities of Research Excellence in Spinal Cord Injury” contract to support independent replication of published studies that could be considered for eventual clinical testing. Recent studies have reported that selective inhibition of the P2X7 receptor improves both the functional and histopathological consequences of a contusive spinal cord injury (SCI) in rats. We repeated two published studies reporting the beneficial effects of pyridoxal-5′-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) or Brilliant blue G (BBG) treatment after SCI (Wang et al., 2004 and Peng et al., 2009). Mild thoracic SCI was first produced in Experiment 1 by means of the MASCIS impactor at T10 (height 6.25 mm, weight 10 gm) followed by intraspinal administration of a P2X7 antagonist (2 μl/10mM) after injury. Treatment with PPADS or another highly selective P2X7R antagonist Brilliant Blue G (BBG) (2 μl/02mM) did not improve locomotive (BBB rating scale) over a 7 week period compared to vehicle treated rats. Also, secondary histopathological changes in terms of overall lesion and cavity volume were not significantly different between the PPADS, BBG, and vehicle treated animals. In the second experiment, the systemic administration of BBG (10 or 50 mg/kg, iv) 15 min, 24 and 72 hours after moderate (12.5 mm) SCI failed to significantly improve motor recovery or histopathological outcome over the 6 week observational period. Although we cannot conclude that there will be no long-term beneficial effects in other spinal cord injury models using selective P2X7 receptor antagonists at different doses or treatment durations, we caution researchers that this potentially exciting therapy requires further preclinical investigations before the implementation of clinical trials targeting severe SCI patients. PMID:22078760

  11. A reassessment of P2X7 receptor inhibition as a neuroprotective strategy in rat models of contusion injury.

    PubMed

    Marcillo, Alexander; Frydel, Beata; Bramlett, Helen M; Dietrich, W Dalton

    2012-02-01

    These experiments were completed as part of an NIH "Facilities of Research Excellence in Spinal Cord Injury" contract to support independent replication of published studies that could be considered for eventual clinical testing. Recent studies have reported that selective inhibition of the P2X7 receptor improves both the functional and histopathological consequences of a contusive spinal cord injury (SCI) in rats. We repeated two published studies reporting the beneficial effects of pyridoxal-5'-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) or Brilliant blue G (BBG) treatment after SCI (Wang et al., 2004 and Peng et al., 2009). Mild thoracic SCI was first produced in Experiment 1 by means of the MASCIS impactor at T10 (height 6.25 mm, weight 10 g) followed by intraspinal administration of a P2X7 antagonist (2 μl/10 mM) after injury. Treatment with PPADS or another highly selective P2X7R antagonist Brilliant Blue G (BBG) (2 μl/02 mM) did not improve locomotive (BBB rating scale) over a 7 week period compared to vehicle treated rats. Also, secondary histopathological changes in terms of overall lesion and cavity volume were not significantly different between the PPADS, BBG, and vehicle treated animals. In the second experiment, the systemic administration of BBG (10 or 50 mg/kg, iv) 15 min, 24 and 72 h after moderate (12.5 mm) SCI failed to significantly improve motor recovery or histopathological outcome over the 6 week observational period. Although we cannot conclude that there will be no long-term beneficial effects in other spinal cord injury models using selective P2X7 receptor antagonists at different doses or treatment durations, we caution researchers that this potentially exciting therapy requires further preclinical investigations before the implementation of clinical trials targeting severe SCI patients. PMID:22078760

  12. Paroxetine suppresses recombinant human P2X7 responses.

    PubMed

    Dao-Ung, Phuong; Skarratt, Kristen K; Fuller, Stephen J; Stokes, Leanne

    2015-12-01

    P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC(50) of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC(50) of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine.

  13. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

    PubMed

    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  14. 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

  15. P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats.

    PubMed

    Zhao, Hengli; Zhang, Xuan; Dai, Zhiqiang; Feng, Yang; Li, Qiang; Zhang, John H; Liu, Xin; Chen, Yujie; Feng, Hua

    2016-01-01

    Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins' expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation. PMID:26980524

  16. P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats

    PubMed Central

    Zhao, Hengli; Zhang, Xuan; Dai, Zhiqiang; Feng, Yang; Li, Qiang; Zhang, John H.; Liu, Xin; Chen, Yujie; Feng, Hua

    2016-01-01

    Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins’ expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation. PMID:26980524

  17. Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death

    PubMed Central

    Xue, Ying; Xie, Yuting; Xue, Bo; Guan, Huaijin

    2016-01-01

    P2X7 receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death. PMID:27738636

  18. 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

  19. P2X7 receptor blockade protects against cisplatin-induced nephrotoxicity in mice by decreasing the activities of inflammasome components, oxidative stress and caspase-3

    SciTech Connect

    Zhang, Yuanyuan; Yuan, Fahuan; Cao, Xuejiao; Zhai, Zhifang; Gang Huang; Du, Xiang; Wang, Yiqin; Zhang, Jingbo; Huang, Yunjian; Zhao, Jinghong; Hou, Weiping

    2014-11-15

    Nephrotoxicity is a common complication of cisplatin chemotherapy and thus limits the use of cisplatin in clinic. The purinergic 2X7 receptor (P2X7R) plays important roles in inflammation and apoptosis in some inflammatory diseases; however, its roles in cisplatin-induced nephrotoxicity remain unclear. In this study, we first assessed the expression of P2X7R in cisplatin-induced nephrotoxicity in C57BL/6 mice, and then we investigated the changes of renal function, histological injury, inflammatory response, and apoptosis in renal tissues after P2X7R blockade in vivo using an antagonist A-438079. Moreover, we measured the changes of nod-like receptor family, pyrin domain containing proteins (NLRP3) inflammasome components, oxidative stress, and proapoptotic genes in renal tissues in cisplatin-induced nephrotoxicity after treatment with A-438079. We found that the expression of P2X7R was significantly upregulated in the renal tubular epithelial cells in cisplatin-induced nephrotoxicity compared with that of the normal control group. Furthermore, pretreatment with A-438079 markedly attenuated the cisplatin-induced renal injury while lightening the histological damage, inflammatory response and apoptosis in renal tissue, and improved the renal function. These effects were associated with the significantly reduced levels of NLRP3 inflammasome components, oxidative stress, p53 and caspase-3 in renal tissues in cisplatin-induced nephrotoxicity. In conclusions, our studies suggest that the upregulated activity of P2X7R might play important roles in the development of cisplatin-induced nephrotoxicity, and P2X7R blockade might become an effective therapeutic strategy for this disease. - Highlights: • The P2X7R expression was markedly upregulated in cisplatin-induced nephrotoxicity. • P2X7R blockade significantly attenuated the cisplatin-induced renal injury. • P2X7R blockade reduced activities of NLRP3 inflammasome components in renal tissue. • P2X7R blockade

  20. 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

  1. 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

  2. Pulmonary Infection with Hypervirulent Mycobacteria Reveals a Crucial Role for the P2X7 Receptor in Aggressive Forms of Tuberculosis

    PubMed Central

    Amaral, Eduardo P.; Ribeiro, Simone C. M.; Lanes, Verônica R.; Almeida, Fabrício M.; de Andrade, Marcelle R. M.; Bomfim, Caio Cesar Barbosa; Salles, Érika M.; Bortoluci, Karina R.; Coutinho-Silva, Robson; Hirata, Mario H.; Alvarez, José M.; Lasunskaia, Elena B.; D'Império-Lima, Maria Regina

    2014-01-01

    The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R−/− mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis. PMID:24991816

  3. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients.

    PubMed

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-10-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94-152.96) and 65.65 months (95% CI, 52.95-76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis.

  4. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients

    PubMed Central

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-01-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94–152.96) and 65.65 months (95% CI, 52.95–76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis. PMID:26337470

  5. Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets.

    PubMed

    Cortés-Garcia, Juan D; López-López, Cintya; Cortez-Espinosa, Nancy; García-Hernández, Mariana H; Guzmán-Flores, Juan M; Layseca-Espinosa, Esther; Portales-Cervantes, Liliana; Portales-Pérez, Diana P

    2016-01-01

    Regulatory T cells that express CD39 (CD39+ Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39+ Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39+ Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25+ CD39+ T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39+ Treg cells and higher levels of ART1 expression in CD4+ CD39+ T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39+ Treg or CD39- Treg cells was observed by 1mM ATP or 60μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39+ Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD.

  6. The effects of early life lead exposure on the expression of P2X7 receptor and synaptophysin in the hippocampus of mouse pups.

    PubMed

    Li, Ning; Zhang, Pingan; Qiao, Mingwu; Shao, Jianfeng; Li, Haozhe; Xie, Wei

    2015-04-01

    The present study was undertaken to investigate the effects of maternal lead exposure on expression of P2X7 receptor and synaptophysin in the hippocampus of mice offspring. Lead exposure initiated from beginning of gestation to weaning. Lead acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1%, 0.5% and 1% groups, respectively. On the 21st postnatal day, the Pb levels were also determined by graphite furnace atomic absorption spectrometry. The expression of P2X7 receptor and synaptophysin in hippocampus was examined by immunohistochemistry and Western blotting. The lead levels in blood and hippocampus of all lead exposure groups were significantly higher than that of the control group (P<0.05). Compared with the control group, the expression of P2X7 receptor was increased in lead exposed groups (P<0.05), but the expression of synaptophysin was decreased (P<0.05). The high expression of P2X7 receptor and low expression of synaptophysin in the hippocampus of pups may contribute to the neurotoxicity associated with maternal Pb exposure.

  7. Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection.

    PubMed

    Wu, C; Zhao, Y; Xiao, X; Fan, Y; Kloc, M; Liu, W; Ghobrial, R M; Lan, P; He, X; Li, X C

    2016-09-01

    Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4-Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft-infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)-gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose-dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4-Ig treatment, inhibited graft-infiltrating M2 cells, prevented transplant vasculopathy, and induced long-term heart allografts survival. These findings highlight the importance of the P2x7r-M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection. PMID:27575724

  8. Interactions of pannexin 1 with NMDA and P2X7 receptors in central nervous system pathologies: Possible role on chronic pain.

    PubMed

    Bravo, D; Maturana, C J; Pelissier, T; Hernández, A; Constandil, L

    2015-11-01

    Pannexin 1 (Panx1) is a glycoprotein that acts as a membrane channel in a wide variety of tissues in mammals. In the central nervous system (CNS) Panx1 is expressed in neurons, astrocytes and microglia, participating in the pathophysiology of some CNS diseases, such as epilepsy, anoxic depolarization after stroke and neuroinflammation. In these conditions Panx1 acts as an important modulator of the neuroinflammatory response, by secreting ATP, by interacting with the P2X7 receptor (P2X7R), and as an amplifier of NMDA receptor (NMDAR) currents, particularly in conditions of pathological neuronal hyperexcitability. Here, we briefly reviewed the current evidences that support the interaction of Panx1 with NMDAR and P2X7R in pathological contexts of the CNS, with special focus in recent data supporting that Panx1 is involved in chronic pain signaling by interacting with NMDAR in neurons and with P2X7R in glia. The participation of Panx1 in chronic pain constitutes a novel topic for research in the field of clinical neurosciences and a potential target for pharmacological interventions in chronic pain. PMID:26211949

  9. Quantitative structure-activity relationship study of P2X7 receptor inhibitors using combination of principal component analysis and artificial intelligence methods.

    PubMed

    Ahmadi, Mehdi; Shahlaei, Mohsen

    2015-01-01

    P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure-activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC's to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7-7-1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure-activity relationship model suggested is robust and satisfactory. PMID:26600858

  10. Quantitative structure-activity relationship study of P2X7 receptor inhibitors using combination of principal component analysis and artificial intelligence methods.

    PubMed

    Ahmadi, Mehdi; Shahlaei, Mohsen

    2015-01-01

    P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure-activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC's to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7-7-1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure-activity relationship model suggested is robust and satisfactory.

  11. Quantitative structure–activity relationship study of P2X7 receptor inhibitors using combination of principal component analysis and artificial intelligence methods

    PubMed Central

    Ahmadi, Mehdi; Shahlaei, Mohsen

    2015-01-01

    P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure–activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC's to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7−7−1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure–activity relationship model suggested is robust and satisfactory. PMID:26600858

  12. P2X7 receptor inhibition increases CNTF in the subventricular zone, but not neurogenesis or neuroprotection after stroke in adult mice.

    PubMed

    Kang, Seong Su; Keasey, Matthew Phillip; Hagg, Theo

    2013-10-01

    Increasing endogenous ciliary neurotrophic factor (CNTF) expression with a pharmacological agent might be beneficial after stroke as CNTF both promotes neurogenesis and, separately, is neuroprotective. P2X7 purinergic receptor inhibition is neuroprotective in rats and increases CNTF release in rat CMT1A Schwann cells. We, first, investigated the role of P2X7 in regulating CNTF and neurogenesis in adult mouse subventricular zone (SVZ). CNTF expression was increased by daily intravenous injections of the P2X7 antagonist Brilliant Blue G (BBG) in naïve C57BL/6 or Balb/c mice over 3 days. Despite the ∼40-60 % increase or decrease in CNTF with BBG or the agonist BzATP, respectively, the number of proliferated BrdU+SVZ nuclei did not change. BBG failed to increase FGF2, which is involved in CNTF-regulated neurogenesis, but induced IL-6, LIF, and EGF, which are known to reduce SVZ proliferation. Injections of IL-6 next to the SVZ induced CNTF and FGF2, but not proliferation, suggesting that IL-6 counteracts their neurogenesis-inducing effects. Following ischemic injury of the striatum by middle cerebral artery occlusion (MCAO), a 3-day BBG treatment increased CNTF in the medial penumbra containing the SVZ. BBG also induced CNTF and LIF, which are known to be protective following stroke, in the whole striatum after MCAO, but not GDNF or BDNF. However, BBG treatment did not reduce the lesion area or apoptosis in the penumbra. Even so, this study shows that P2X7 can be targeted with systemic drug treatments to differentially regulate neurotrophic factors in the brain following stroke.

  13. P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART

    PubMed Central

    Menkova-Garnier, Inna; Hocini, Hakim; Foucat, Emile; Tisserand, Pascaline; Bourdery, Laure; Delaugerre, Constance; Benne, Clarisse; Lévy, Yves; Lelièvre, Jean-Daniel

    2016-01-01

    Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an in vitro system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (Fas, P2X7, CD39/CD73). P2X7 expression was abnormally strong and there was no CD73 mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that in vitro inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs. PMID:27082982

  14. P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART.

    PubMed

    Menkova-Garnier, Inna; Hocini, Hakim; Foucat, Emile; Tisserand, Pascaline; Bourdery, Laure; Delaugerre, Constance; Benne, Clarisse; Lévy, Yves; Lelièvre, Jean-Daniel

    2016-04-01

    Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an in vitro system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (Fas, P2X7, CD39/CD73). P2X7 expression was abnormally strong and there was no CD73 mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that in vitro inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs. PMID:27082982

  15. Mechanism of microglia neuroprotection: Involvement of P2X7, TNFα, and valproic acid.

    PubMed

    Masuch, Annette; Shieh, Chu-Hsin; van Rooijen, Nico; van Calker, Dietrich; Biber, Knut

    2016-01-01

    Recently, we have demonstrated that ramified microglia are neuroprotective in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in organotypic hippocampal slice cultures (OHSCs). The present study aimed to elucidate the underlying neuron-glia communication mechanism. It is shown here that pretreatment of OHSC with high concentrations of adenosine 5'-triphosphate (ATP) reduced NMDA-induced neuronal death only in presence of microglia. Specific agonists and antagonists identified the P2X7 receptor as neuroprotective receptor which was confirmed by absence of ATP-dependent neuroprotection in P2X7-deficient OHSC. Microglia replenished chimeric OHSC consisting of wild-type tissue replenished with P2X7-deficient microglia confirmed the involvement of microglial P2X7 receptor in neuroprotection. Stimulation of P2X7 in primary microglia induced tumor necrosis factor α (TNFα) release and blocking TNFα by a neutralizing antibody in OHSC abolished neuroprotection by ATP. OHSC from TNFα-deficient mice show increased exicitoxicity and activation of P2X7 did not rescue neuronal survival in the absence of TNFα. The neuroprotective effect of valproic acid (VPA) was strictly dependent on the presence of microglia and was mediated by upregulation of P2X7 in the cells. The present study demonstrates that microglia-mediated neuroprotection depends on ATP-activated purine receptor P2X7 and induction of TNFα release. This neuroprotective pathway was strengthened by VPA elucidating a novel mechanism for the neuroprotective function of VPA.

  16. Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

    PubMed Central

    Melo, A C; Moeller, P D; Glasgow, H; Burkholder, J M; Ramsdell, J S

    2001-01-01

    Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP. PMID:11677182

  17. Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice.

    PubMed

    Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E; Alkana, Ronald L; Tsukamoto, Hidekazu; Davies, Daryl L

    2015-08-15

    The present investigation tested the role of ATP-activated P2X7 receptors (P2X7Rs) in alcohol-induced brain damage using a model that combines intragastric (iG) ethanol feeding and high fat diet in C57BL/6J mice (Hybrid). The Hybrid paradigm caused increased levels of pro-inflammatory markers, changes in microglia and astrocytes, reduced levels of neuronal marker NeuN and increased P2X7R expression in ethanol-sensitive brain regions. Observed changes in P2X7R and NeuN expression were more pronounced in Hybrid paradigm with inclusion of additional weekly binges. In addition, high fat diet during Hybrid exposure aggravated the increase in P2X7R expression and activation of glial cells.

  18. Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice.

    PubMed

    Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E; Alkana, Ronald L; Tsukamoto, Hidekazu; Davies, Daryl L

    2015-08-15

    The present investigation tested the role of ATP-activated P2X7 receptors (P2X7Rs) in alcohol-induced brain damage using a model that combines intragastric (iG) ethanol feeding and high fat diet in C57BL/6J mice (Hybrid). The Hybrid paradigm caused increased levels of pro-inflammatory markers, changes in microglia and astrocytes, reduced levels of neuronal marker NeuN and increased P2X7R expression in ethanol-sensitive brain regions. Observed changes in P2X7R and NeuN expression were more pronounced in Hybrid paradigm with inclusion of additional weekly binges. In addition, high fat diet during Hybrid exposure aggravated the increase in P2X7R expression and activation of glial cells. PMID:26198936

  19. Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice

    PubMed Central

    Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E; Alkana, Ronald L; Tsukamoto, Hidekatsu; Davies, Daryl L.

    2015-01-01

    The present investigation tested the role of ATP-activated P2X7 receptors (P2X7Rs) in alcohol-induced brain damage using a model that combines intragastric (iG) ethanol feeding and high fat diet in C57BL/6J mice (Hybrid). The Hybrid paradigm caused increased levels of pro-inflammatory markers, changes in microglia and astrocytes, reduced levels of neuronal marker NeuN and increased P2X7R expression in ethanol-sensitive brain regions. Observed changes in P2X7R and NeuN expression were more pronounced in Hybrid paradigm with inclusion of additional weekly binges. In addition, high fat diet during Hybrid exposure aggravated the increase in P2X7R expression and activation of glial cells. PMID:26198936

  20. 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

  1. A consensus segment in the M2 domain of the hP2X(7) receptor shows ion channel activity in planar lipid bilayers and in biological membranes.

    PubMed

    de Souza, Cristina Alves Magalhães; Teixeira, Pedro Celso Nogueira; Faria, Robson Xavier; Krylova, Oxana; Pohl, Peter; Alves, Luiz Anastacio

    2012-01-01

    The P2X(7) receptor (P2X(7)R) is an ATP-gated, cation-selective channel permeable to Na(+), K(+) and Ca(2+). This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X(7)R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X(7)R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X(7)R. The conserved part of the M2 domain of P2X(7)R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.

  2. Constructing an atomic-resolution model of human P2X7 receptor followed by pharmacophore modeling to identify potential inhibitors.

    PubMed

    Ahmadi, Mehdi; Nowroozi, Amin; Shahlaei, Mohsen

    2015-09-01

    The P2X purinoceptor 7 (P2X7R) is a trimeric ATP-activated ion channel gated by extracellular ATP. P2X7R has important role in numerous diseases including pain, neurodegeneration, and inflammatory diseases such as rheumatoid arthritis and osteoarthritis. In this prospective, the discovery of small-molecule inhibitors for P2X7R as a novel therapeutic target has received considerable attention in recent years. At first, 3D structure of P2X7R was built by using homology modeling (HM) and a 50ns molecular dynamics simulation (MDS). Ligand-based quantitative pharmacophore modeling methodology of P2X7R antagonists were developed based on training set of 49 compounds. The best four-feature pharmacophore model, includes two hydrophobic aromatic, one hydrophobic and one aromatic ring features, has the highest correlation coefficient (0.874), cost difference (368.677), low RMSD (2.876), as well as it shows a high goodness of fit and enrichment factor. Consequently, some hit compounds were introduced as final candidates by employing virtual screening and molecular docking procedure simultaneously. Among these compounds, six potential molecule were identified as potential virtual leads which, as such or upon further optimization, can be used to design novel P2X7R inhibitors.

  3. Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

    PubMed Central

    Li, Shuo; Li, Xuejing; Coddou, Claudio; Geng, Xuyun; Wei, Junli; Sun, Jinsheng

    2014-01-01

    ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression. PMID:24796752

  4. Involvement of the P2X7-NLRP3 axis in leukemic cell proliferation and death

    PubMed Central

    Salaro, Erica; Rambaldi, Alessia; Falzoni, Simonetta; Amoroso, Francesca Saveria; Franceschini, Alessia; Sarti, Alba Clara; Bonora, Massimo; Cavazzini, Francesco; Rigolin, Gian Matteo; Ciccone, Maria; Audrito, Valentina; Deaglio, Silvia; Pelegrin, Pablo; Pinton, Paolo; Cuneo, Antonio; Di Virgilio, Francesco

    2016-01-01

    Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R, ASC and NLRP3 were investigated by Western blot, PCR and transfection techniques. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 was dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. The P2X7R was also up-modulated in hematopoietic cells from NLRP3-KO mice. In conclusion, we show that NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL. PMID:27221966

  5. Involvement of the P2X7-NLRP3 axis in leukemic cell proliferation and death.

    PubMed

    Salaro, Erica; Rambaldi, Alessia; Falzoni, Simonetta; Amoroso, Francesca Saveria; Franceschini, Alessia; Sarti, Alba Clara; Bonora, Massimo; Cavazzini, Francesco; Rigolin, Gian Matteo; Ciccone, Maria; Audrito, Valentina; Deaglio, Silvia; Pelegrin, Pablo; Pinton, Paolo; Cuneo, Antonio; Di Virgilio, Francesco

    2016-01-01

    Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R, ASC and NLRP3 were investigated by Western blot, PCR and transfection techniques. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 was dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. The P2X7R was also up-modulated in hematopoietic cells from NLRP3-KO mice. In conclusion, we show that NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL. PMID:27221966

  6. The absence of P2X7 receptors (P2rx7) on non-haematopoietic cells leads to selective alteration in mood-related behaviour with dysregulated gene expression and stress reactivity in mice.

    PubMed

    Csölle, Cecilia; Andó, Rómeó D; Kittel, Ágnes; Gölöncsér, Flóra; Baranyi, Mária; Soproni, Krisztina; Zelena, Dóra; Haller, József; Németh, Tamás; Mócsai, Attila; Sperlágh, Beáta

    2013-02-01

    The purpose of this study was to explore how genetic deletion and pharmacological antagonism of the P2X7 receptor (P2rx7) alter mood-related behaviour, gene expression and stress reactivity in the brain. The forced swim test (FST), tail suspension test (TST) and amphetamine-induced hyperlocomotion (AH) tests were used in wild-type (P2rx7(+/+)) and P2rx7-deficient (P2rx7(-/-)) mice. Biogenic amine levels were analysed in the amygdala and striatum, adrenocorticotropic hormone (ACTH) and corticosterone levels were measured in the plasma and pituitary after restraint stress. Chimeric mice were generated by bone marrow transplantation. A whole genome microarray analysis with real-time polymerase chain reaction validation was performed on the amygdala. In the absence of P2rx7s decreased behavioural despair in the FST, reduced immobility in the TST and attenuated amphetamine-induced hyperactivity were detected. Basal norepinephrine levels were elevated in the amygdala, whereas stress-induced ACTH and corticosterone responses were alleviated in P2rx7(-/-) mice. Sub-acute treatment with the selective P2rx7 antagonist, Brilliant Blue G, reproduced the effect of genetic deletion in the TST and AH test in P2rx7(+/+) but not P2rx7(-/-) mice. No change in behavioural phenotype was observed in chimeras lacking the P2rx7 in their haematopoietic compartment. Whole genome microarray analysis indicated a widespread up- and down-regulation of genes crucial for synaptic function and neuroplasticity by genetic deletion. Here, we present evidence that the absence of P2rx7s on non-haematopoietic cells leads to a mood-stabilizing phenotype in several behavioural models and suggest a therapeutic potential of P2rx7 antagonists for the treatment of mood disorders.

  7. Pharmacological blockage and P2X7 deletion hinder aversive memories: reversion in an enriched environment.

    PubMed

    Campos, R C; Parfitt, G M; Polese, C E; Coutinho-Silva, R; Morrone, F B; Barros, D M

    2014-11-01

    Adenosine triphosphate (ATP) plays a role in cell signaling. It was soon proposed that ATP activates ionotropic P2X receptors, exerting an influence on neurons as well as on glial cells. In addition to the fact that the activation of P2X and P2Y receptors can stimulate or inhibit the release of glutamate from rat hippocampal neurons, the release of ATP has been implicated in hippocampal long-term potentiation (LTP). Through different behavioral paradigms, this study aimed to investigate the participation of P2X7R in genetically modified (knockout (KO)) mice with the suppressed expression of this receptor and in the pharmacological blockage of this receptor in rats, as well as to evaluate the effect of environmental enrichment on potential mnemonic deficits. The results suggest that P2X7R participates in aversive memory processes: pharmacological blockage with the selective P2X7R antagonist, A-740003, in different time frames elicited dose-dependent impairments in memory acquisition, consolidation and retrieval in rats that were submitted to the contextual fear-conditioning (FC) task, and the deletion of P2X7R hampered the aversive memory processes of mice that were subjected to the FC paradigm. Experiments using mice that were subjected to environmental enrichment suggest that this form of stimulation reverses mnemonic impairments that are ascribed to the absence of the P2X7R, suggesting that these receptors do not participate on such a reversal. Finally, no alterations were observed in the habituation memory of P2X7KO mice.

  8. Pharmacological blockage and P2X7 deletion hinder aversive memories: reversion in an enriched environment.

    PubMed

    Campos, R C; Parfitt, G M; Polese, C E; Coutinho-Silva, R; Morrone, F B; Barros, D M

    2014-11-01

    Adenosine triphosphate (ATP) plays a role in cell signaling. It was soon proposed that ATP activates ionotropic P2X receptors, exerting an influence on neurons as well as on glial cells. In addition to the fact that the activation of P2X and P2Y receptors can stimulate or inhibit the release of glutamate from rat hippocampal neurons, the release of ATP has been implicated in hippocampal long-term potentiation (LTP). Through different behavioral paradigms, this study aimed to investigate the participation of P2X7R in genetically modified (knockout (KO)) mice with the suppressed expression of this receptor and in the pharmacological blockage of this receptor in rats, as well as to evaluate the effect of environmental enrichment on potential mnemonic deficits. The results suggest that P2X7R participates in aversive memory processes: pharmacological blockage with the selective P2X7R antagonist, A-740003, in different time frames elicited dose-dependent impairments in memory acquisition, consolidation and retrieval in rats that were submitted to the contextual fear-conditioning (FC) task, and the deletion of P2X7R hampered the aversive memory processes of mice that were subjected to the FC paradigm. Experiments using mice that were subjected to environmental enrichment suggest that this form of stimulation reverses mnemonic impairments that are ascribed to the absence of the P2X7R, suggesting that these receptors do not participate on such a reversal. Finally, no alterations were observed in the habituation memory of P2X7KO mice. PMID:25239372

  9. An Improved Method for P2X7R Antagonist Screening

    PubMed Central

    Soares-Bezerra, Rômulo José; Ferreira, Natiele Carla da Silva; Alberto, Anael Viana Pinto; Bonavita, André Gustavo; Fidalgo-Neto, Antônio Augusto; Calheiros, Andrea Surrage; Frutuoso, Valber da Silva; Alves, Luiz Anastacio

    2015-01-01

    ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist PMID:25993132

  10. 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

  11. microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus

    PubMed Central

    Jimenez-Mateos, Eva M.; Arribas-Blazquez, Marina; Sanz-Rodriguez, Amaya; Concannon, Caoimhin; Olivos-Ore, Luis A.; Reschke, Cristina R.; Mooney, Claire M.; Mooney, Catherine; Lugara, Eleonora; Morgan, James; Langa, Elena; Jimenez-Pacheco, Alba; Silva, Luiz Fernando Almeida; Mesuret, Guillaume; Boison, Detlev; Miras-Portugal, M. Teresa; Letavic, Michael; Artalejo, Antonio R.; Bhattacharya, Anindya; Diaz-Hernandez, Miguel; Henshall, David C.; Engel, Tobias

    2015-01-01

    The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7−/− mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain. PMID:26631939

  12. The P2X7/P2X4 interaction shapes the purinergic response in murine macrophages.

    PubMed

    Pérez-Flores, Gabriela; Lévesque, Sébastien A; Pacheco, Jonathan; Vaca, Luis; Lacroix, Steve; Pérez-Cornejo, Patricia; Arreola, Jorge

    2015-11-20

    The ATP-gated P2X4 and P2X7 receptors are cation channels, co-expressed in excitable and non-excitable cells and play important roles in pain, bone development, cytokine release and cell death. Although these receptors interact the interacting domains are unknown and the functional consequences of this interaction remain unclear. Here we show by co-immunoprecipitation that P2X4 interacts with the C-terminus of P2X7 and by fluorescence resonance energy transfer experiments that this receptor-receptor interaction is driven by ATP. Furthermore, disrupting the ATP-driven interaction by knocking-out P2X4R provoked an attenuation of P2X7-induced cell death, dye uptake and IL-1β release in macrophages. Thus, P2X7 interacts with P2X4 via its C-terminus and disrupting the P2X7/P2X4 interaction hinders physiological responses in immune cells.

  13. Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death

    PubMed Central

    Draganov, Dobrin; Gopalakrishna-Pillai, Sailesh; Chen, Yun-Ru; Zuckerman, Neta; Moeller, Sara; Wang, Carrie; Ann, David; Lee, Peter P.

    2015-01-01

    Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca2+/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy. PMID:26552848

  14. Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death.

    PubMed

    Draganov, Dobrin; Gopalakrishna-Pillai, Sailesh; Chen, Yun-Ru; Zuckerman, Neta; Moeller, Sara; Wang, Carrie; Ann, David; Lee, Peter P

    2015-01-01

    Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy. PMID:26552848

  15. P2X7 as a new target for chrysophanol to treat lipopolysaccharide-induced depression in mice.

    PubMed

    Zhang, Kai; Liu, Jingyan; You, Xintong; Kong, Ping; Song, Yichen; Cao, Lu; Yang, Song; Wang, Wenbing; Fu, Qiang; Ma, Zhangqiang

    2016-02-01

    P2X7 receptor is a ligand gated ion channel found peripheral macrophages and microglia in the nervous system. The current study investigated the relationship between the activated P2X7 and depression for the first time. Chrysophanol (Chr) was examined for its protective effects against depression targeting P2X7. Chr (20mg/kg, 40mg/kg) and fluoxetine (20mg/kg) were intragastrically treated once daily for 7 consecutive days. Lipopolysaccharide (LPS, 0.5mg/kg) was intraperitoneally injected to develop depression model 30min after drug administration on day 7. Behavioral tests were measured 24h after LPS injection. Interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α levels in serum and hippocampus were measured by enzyme-linked immunosorbent assay (ELISA). The expressions of P2X7/NF-κB pathway-related proteins were assessed by western blot. The findings showed that Chr remarkably reduced the elevations of IL-6, IL-1β and TNF-α caused by LPS stimulation. The expressions of P2X7, p-IKKα, p-IKKβ, p-IκBα and p-NF-κBp65 were significantly decreased by Chr pretreatment. In addition, immobility time in tail suspension test (TST) and forced swimming test (FST) were reduced by Chr without affecting spontaneous locomotor activity in open filed test (OFT) and the preference for sucrose was also recovered in sucrose preference test (SPT) with Chr preconditioning. Thus, it is reasonable to speculate that Chr might exert antidepressant effect through inhibiting P2X7/NF-κB signaling pathway. PMID:26724370

  16. The microglial ATP-gated ion channel P2X7 as a CNS drug target.

    PubMed

    Bhattacharya, Anindya; Biber, Knut

    2016-10-01

    Based on promising preclinical evidence, microglial P2X7 has increasingly being recognized as a target for therapeutic intervention in neurological and psychiatric diseases. However, despite this knowledge no P2X7-related drug has yet entered clinical trials with respect to CNS diseases. We here discuss the current literature on P2X7 being a drug target and identify unsolved issues and still open questions that have hampered the development of P2X7 dependent therapeutic approaches for CNS diseases. It is concluded here that the lack of brain penetrating P2X7 antagonists is a major obstacle in the field and that central P2X7 is a yet untested clinical drug target. In the CNS, microglial P2X7 activation causes neuroinflammation, which in turn plays a role in various CNS disorders. This has resulted in a surge of brain penetrant P2X7 antagonists. P2X7 is a viable, clinically untested CNS drug target. GLIA 2016;64:1772-1787.

  17. The microglial ATP-gated ion channel P2X7 as a CNS drug target.

    PubMed

    Bhattacharya, Anindya; Biber, Knut

    2016-10-01

    Based on promising preclinical evidence, microglial P2X7 has increasingly being recognized as a target for therapeutic intervention in neurological and psychiatric diseases. However, despite this knowledge no P2X7-related drug has yet entered clinical trials with respect to CNS diseases. We here discuss the current literature on P2X7 being a drug target and identify unsolved issues and still open questions that have hampered the development of P2X7 dependent therapeutic approaches for CNS diseases. It is concluded here that the lack of brain penetrating P2X7 antagonists is a major obstacle in the field and that central P2X7 is a yet untested clinical drug target. In the CNS, microglial P2X7 activation causes neuroinflammation, which in turn plays a role in various CNS disorders. This has resulted in a surge of brain penetrant P2X7 antagonists. P2X7 is a viable, clinically untested CNS drug target. GLIA 2016;64:1772-1787. PMID:27219534

  18. Tolerability and efficacy study of P2X7 inhibition in experimental Charcot-Marie-Tooth type 1A (CMT1A) neuropathy.

    PubMed

    Sociali, Giovanna; Visigalli, Davide; Prukop, Thomas; Cervellini, Ilaria; Mannino, Elena; Venturi, Consuelo; Bruzzone, Santina; Sereda, Michael W; Schenone, Angelo

    2016-11-01

    Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy for which pharmacological treatments are not yet available. An abnormally high intracellular Ca(2+) concentration was observed in Schwann cells (SC) from CMT1A rats, caused by the PMP22-mediated overexpression of the P2X7 purinoceptor. The purpose of this study was to investigate the tolerability and therapeutic potential of a pharmacological antagonist of the P2X7 receptor (A438079) in CMT1A. A438079 ameliorated in vitro myelination of organotypic DRG cultures from CMT1A rats. Furthermore, we performed an experimental therapeutic trial in PMP22 transgenic and in wild-type rats. A preliminary dose-escalation trial showed that 3mg/kg A438079 administered via intraperitoneal injection every 24h for four weeks was well tolerated by wild type and CMT1A rats. Affected rats treated with 3mg/kg A438079 revealed a significant improvement of the muscle strength, when compared to placebo controls. Importantly, histologic analysis revealed a significant increase of the total number of myelinated axons in tibial nerves. Moreover, a significant decrease of the hypermyelination of small caliber axons and a significant increase of the frequency and diameter of large caliber myelinated axons was highlighted. An improved distal motor latencies was recorded, whereas compound muscle action potentials (CMAP) remained unaltered. A438079 reduced the SC differentiation defect in CMT1A rats. These results show that pharmacological inhibition of the P2X7 receptor is well tolerated in CMT1A rats and represents a proof-of-principle that antagonizing this pathway may correct the molecular derangements and improve the clinical phenotype in the CMT1A neuropathy. PMID:27431093

  19. Tolerability and efficacy study of P2X7 inhibition in experimental Charcot-Marie-Tooth type 1A (CMT1A) neuropathy.

    PubMed

    Sociali, Giovanna; Visigalli, Davide; Prukop, Thomas; Cervellini, Ilaria; Mannino, Elena; Venturi, Consuelo; Bruzzone, Santina; Sereda, Michael W; Schenone, Angelo

    2016-11-01

    Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy for which pharmacological treatments are not yet available. An abnormally high intracellular Ca(2+) concentration was observed in Schwann cells (SC) from CMT1A rats, caused by the PMP22-mediated overexpression of the P2X7 purinoceptor. The purpose of this study was to investigate the tolerability and therapeutic potential of a pharmacological antagonist of the P2X7 receptor (A438079) in CMT1A. A438079 ameliorated in vitro myelination of organotypic DRG cultures from CMT1A rats. Furthermore, we performed an experimental therapeutic trial in PMP22 transgenic and in wild-type rats. A preliminary dose-escalation trial showed that 3mg/kg A438079 administered via intraperitoneal injection every 24h for four weeks was well tolerated by wild type and CMT1A rats. Affected rats treated with 3mg/kg A438079 revealed a significant improvement of the muscle strength, when compared to placebo controls. Importantly, histologic analysis revealed a significant increase of the total number of myelinated axons in tibial nerves. Moreover, a significant decrease of the hypermyelination of small caliber axons and a significant increase of the frequency and diameter of large caliber myelinated axons was highlighted. An improved distal motor latencies was recorded, whereas compound muscle action potentials (CMAP) remained unaltered. A438079 reduced the SC differentiation defect in CMT1A rats. These results show that pharmacological inhibition of the P2X7 receptor is well tolerated in CMT1A rats and represents a proof-of-principle that antagonizing this pathway may correct the molecular derangements and improve the clinical phenotype in the CMT1A neuropathy.

  20. 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

  1. 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.

  2. Regulation of the P2X7R by microRNA-216b in human breast cancer

    SciTech Connect

    Zheng, Luming; Zhang, Xukui; Yang, Feng; Zhu, Jian; Zhou, Peng; Yu, Fang; Hou, Lei; Xiao, Lei; He, Qingqing; Wang, Baocheng

    2014-09-12

    Highlights: • We suggest the expression level of miR-216b and P2X7R in breast cancer tissues and cell lines. • We demonstrated that miR-216b directly targets and inhibits P2X7R. • We suggested miR-216b can attenuate ATP/P2X7R signaling pathways and induced Bcl-2/caspase-3 pathway. - Abstract: Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that P2X7R was up-regulated and miR-216b was down-regulated in breast cancer cell lines and tissues. Using bioinformatic analysis and 3′UTR luciferase reporter assay, we determined P2X7R can be directly targeted by miR-216b, which can down-regulate endogenous P2X7R mRNA and protein levels. Ectopic expression of miR-216b mimics leads to inhibited cell growth and apoptosis, while blocking expression of the miR-216b results in increased cell proliferation. Furthermore, our findings demonstrate that knockdown of P2X7R promotes apoptosis in breast cancer cells through down-regulating Bcl-2 and increasing the cleavage caspase-3 protein level. Finally, we confirmed that down-regulation of miR-216b in breast cancer is inversely associated with P2X7R expression level. Together, these findings establish miR-216b as a novel regulator of P2X7R and a potential therapeutic target for breast cancer.

  3. A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis.

    PubMed

    Gu, Ben J; Field, Judith; Dutertre, Sébastien; Ou, Amber; Kilpatrick, Trevor J; Lechner-Scott, Jeannette; Scott, Rodney; Lea, Rodney; Taylor, Bruce V; Stankovich, Jim; Butzkueven, Helmut; Gresle, Melissa; Laws, Simon M; Petrou, Steven; Hoffjan, Sabine; Akkad, Denis A; Graham, Colin A; Hawkins, Stanley; Glaser, Anna; Bedri, Sahl Khalid; Hillert, Jan; Matute, Carlos; Antiguedad, Alfredo; Wiley, James S

    2015-10-01

    Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.

  4. P2X7 and NRAMP1/SLC11 A1 gene polymorphisms in Mexican mestizo patients with pulmonary tuberculosis

    PubMed Central

    Niño-Moreno, P; Portales-Pérez, D; Hernández-Castro, B; Portales-Cervantes, L; Flores-Meraz, V; Baranda, L; Gómez-Gómez, A; Acuña-Alonzo, V; Granados, J; González-Amaro, R

    2007-01-01

    Tuberculosis remains one of the most important infectious diseases worldwide. Several studies have suggested that genetic factors may affect susceptibility to tuberculosis, but the specific genes involved have not yet been fully characterized. NRAMP1/SLC11 A1 and P2X7 genes have been linked to increased risk for tuberculosis in some African and Asiatic populations. To explore the potential role of these genes in the susceptibility to pulmonary tuberculosis in a Mexican mestizo population, we evaluated the association of D543N and 3′-UTR polymorphisms in NRAMP1/SLC11 A1 and − 762 and A1513C polymorphisms in P2X7 genes with the risk for tuberculosis. Polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction fragment length polymorphism analysis, and allelic-specific PCR was employed. We found no significant differences in allelic frequency in NRAMP1/SLC11 A1 gene polymorphisms in 94 patients with tuberculosis compared to 100 healthy contacts. Similarly, no significant association of the P2X7−762 gene polymorphism with tuberculosis was detected. In contrast, the P2X7 A1513C polymorphism was associated significantly with tuberculosis (P= 0·02, odds ratio = 5·28, 95% CI, 0·99–37·69), an association that had not been reported previously. However, when the function of P2X7 was assessed by an l-selectin loss assay, we did not find significant differences in patients compared to healthy contacts or between PPD+ and PPD– control individuals. This study further supports the complex role of P2X7 gene in host regulation of Mycobacterium tuberculosis infection, and demonstrates that different associations of gene polymorphisms and tuberculosis are found in distinct racial populations. PMID:17493019

  5. P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes.

    PubMed

    Seref-Ferlengez, Zeynep; Maung, Stephanie; Schaffler, Mitchell B; Spray, David C; Suadicani, Sylvia O; Thi, Mia M

    2016-01-01

    Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood. We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. To simulate in vitro the glucose levels to which bone cells are exposed in healthy vs. diabetic bones, we cultured osteoblast and osteocyte cell lines for 10 days in medium containing 5.5 or 25 mM glucose. High glucose effects on expression and function of P2Rs and Panx1 channels were determined by Western Blot analysis, quantification of Ca2+ responses to P2R agonists and oscillatory fluid shear stress (± 10 dyne/cm2), and measurement of flow-induced ATP release. Diabetic C57BL/6J-Ins2Akita mice were used to evaluate in vivo effects of high glucose on P2R and Panx1. Western blotting indicated altered P2X7R, P2Y2R and P2Y4R expression in high glucose exposed bone cells, and in diabetic bone tissue. Moreover, high glucose blunted normal P2R- and flow-induced Ca2+ signaling and ATP release from osteocytes. These findings indicate that T1D impairs load-induced ATP signaling in osteocytes and affects osteoblast function, which are essential for maintaining bone health. PMID:27159053

  6. P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    PubMed Central

    Maung, Stephanie; Schaffler, Mitchell B.; Spray, David C.; Suadicani, Sylvia O.; Thi, Mia M.

    2016-01-01

    Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood. We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. To simulate in vitro the glucose levels to which bone cells are exposed in healthy vs. diabetic bones, we cultured osteoblast and osteocyte cell lines for 10 days in medium containing 5.5 or 25 mM glucose. High glucose effects on expression and function of P2Rs and Panx1 channels were determined by Western Blot analysis, quantification of Ca2+ responses to P2R agonists and oscillatory fluid shear stress (± 10 dyne/cm2), and measurement of flow-induced ATP release. Diabetic C57BL/6J-Ins2Akita mice were used to evaluate in vivo effects of high glucose on P2R and Panx1. Western blotting indicated altered P2X7R, P2Y2R and P2Y4R expression in high glucose exposed bone cells, and in diabetic bone tissue. Moreover, high glucose blunted normal P2R- and flow-induced Ca2+ signaling and ATP release from osteocytes. These findings indicate that T1D impairs load-induced ATP signaling in osteocytes and affects osteoblast function, which are essential for maintaining bone health. PMID:27159053

  7. 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

  8. Genetic Association for P2X7R rs3751142 and CARD8 rs2043211 Polymorphisms for Susceptibility of Gout in Korean Men: Multi-Center Study

    PubMed Central

    2016-01-01

    The aim of this study was to determine the association between P2X7R rs3751142 and CARD8 rs2043211 polymorphisms and gout susceptibility in male Korean subjects. This study enrolled a total of 242 male patients with gout and 280 healthy controls. The polymorphisms of two individual genes including rs3751142(C>A) in the P2X7R gene and rs2043211(A>T) in the CARD8 gene were assessed using Taq-Man analysis. Statistical analyses were performed using the Chi-square test, Kruskal-Wallis test, and logistic regression analyses. A difference in genotypic frequency of the P2X7R rs3751142 and CARD8 rs2043211 genes was not detected between gout and control patients. Clinical parameters including age, onset age, disease duration, body mass index, and serum uric acid levels were not different among the three genotypes for either P2X7R or CARD8 (P > 0.05 for all). A pair-wise comparison of P2X7R rs3751142 and CARD8 rs2043211 genotype combinations revealed that subjects with the CA P2X7R rs3751142 genotype and the TT CARD8 rs2043211 genotype had a trend toward a higher risk of gout compared to the CC/AA combination (P = 0.056, OR = 2.618, 95% CI 0.975 - 7.031). In conclusion, this study revealed that genetic variability of the P2X7R rs3751142 and CARD8 rs2043211 genes might, in part, be associated with susceptibility for gout. PMID:27550484

  9. Genetic Association for P2X7R rs3751142 and CARD8 rs2043211 Polymorphisms for Susceptibility of Gout in Korean Men: Multi-Center Study.

    PubMed

    Lee, Sung Won; Lee, Shin Seok; Oh, Dong Ho; Park, Dong Jin; Kim, Hyun Sook; Choi, Jung Ran; Chae, Soo Cheon; Yun, Ki Jung; Chung, Won Tae; Choe, Jung Yoon; Kim, Seong Kyu

    2016-10-01

    The aim of this study was to determine the association between P2X7R rs3751142 and CARD8 rs2043211 polymorphisms and gout susceptibility in male Korean subjects. This study enrolled a total of 242 male patients with gout and 280 healthy controls. The polymorphisms of two individual genes including rs3751142(C>A) in the P2X7R gene and rs2043211(A>T) in the CARD8 gene were assessed using Taq-Man analysis. Statistical analyses were performed using the Chi-square test, Kruskal-Wallis test, and logistic regression analyses. A difference in genotypic frequency of the P2X7R rs3751142 and CARD8 rs2043211 genes was not detected between gout and control patients. Clinical parameters including age, onset age, disease duration, body mass index, and serum uric acid levels were not different among the three genotypes for either P2X7R or CARD8 (P > 0.05 for all). A pair-wise comparison of P2X7R rs3751142 and CARD8 rs2043211 genotype combinations revealed that subjects with the CA P2X7R rs3751142 genotype and the TT CARD8 rs2043211 genotype had a trend toward a higher risk of gout compared to the CC/AA combination (P = 0.056, OR = 2.618, 95% CI 0.975 - 7.031). In conclusion, this study revealed that genetic variability of the P2X7R rs3751142 and CARD8 rs2043211 genes might, in part, be associated with susceptibility for gout. PMID:27550484

  10. Astrocyte-mediated ischemic tolerance.

    PubMed

    Hirayama, Yuri; Ikeda-Matsuo, Yuri; Notomi, Shoji; Enaida, Hiroshi; Kinouchi, Hiroyuki; Koizumi, Schuichi

    2015-03-01

    Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential. PMID:25740510

  11. Cardioprotective Effects of Genistin in Rat Myocardial Ischemia-Reperfusion Injury Studies by Regulation of P2X7/NF-κB Pathway

    PubMed Central

    Gu, Meng; Zheng, Ai-bin; Jin, Jing; Cui, Yue; Zhang, Ning; Che, Zhi-ping; Wang, Yan; Zhan, Jie; Tu, Wen-juan

    2016-01-01

    The present study aimed to assess the effects and mechanisms of genistin in the rat model of myocardial ischemia reperfusion injury. The rat hearts were exposed to the left anterior descending coronary artery (LAD) ligation for 30 min followed by 1 h of reperfusion. In the rat of myocardial ischemia/reperfusion (MI/R), it was found that genistin pretreatment reduced myocardial infarct size, improved the heart rate, and decreased creatine kinase (CK) and lactate dehydrogenase (LDH) levels in coronary flow. This pretreatment also increased catalase (CAT), superoxide dismutase (SOD) activities but decreased glutathione (GSH), malondialdehyde (MDA) levels. Furthermore, we determined that genistin can ameliorate the impaired mitochondrial morphology and oxidation system; interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) levels were also recovered. Besides, related-proteins of nuclear factor kappa-B (NF-κB) signal pathway activated by P2X7 were investigated to determine the molecular mechanism of genistin and their expressions were measured by western blot. These results presented here demonstrated that genistin enhanced the protective effect on the rats with myocardial ischemia reperfusion injury. Therefore, the cardioprotective effects of genistin may rely on its antioxidant and anti-inflammatory activities via suppression of P2X7/NF-κB pathways. PMID:27087823

  12. The discovery and preclinical characterization of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists.

    PubMed

    Rech, Jason C; Bhattacharya, Anindya; Branstetter, Bryan J; Love, Christopher J; Leenaerts, Joseph E; Cooymans, Ludwig P; Eckert, William A; Ao, Hong; Wang, Qi; Chaplan, Sandra R; Wickenden, Alan D; Lebsack, Alec D; Breitenbucher, J Guy

    2016-10-01

    The synthesis, SAR and preclinical characterization of a series of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists is described herein. The lead compounds are potent inhibitors in Ca(2+) flux and whole blood IL-1β P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1β release in a mouse ex vivo model with a 50mg/kg oral dose. Evaluation of compound 1e in the mouse SNI tactile allodynia, carrageenan-induced paw edema or CIA models resulted in no analgesic or anti-inflammatory effects. PMID:27595421

  13. Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

    SciTech Connect

    Chen, Shayan; Feng, Wenli; Yang, Xiao; Yang, Wanzhu; Ru, Yongxin; Liao, Jinfeng; Wang, Lina; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

    2014-04-18

    Highlights: • We study the impact of leukemic microenvironment on P2X family receptors in Mφs. • Bone marrow and spleen Mφs are studied in Notch1-induced mouse leukemia model. • Increased expression of P2X7R is found in Mφs during the development of leukemia. • Elevated P2X7R-mediated calcium response is found in Mφs at late stage of leukemia. • More apoptotic Mφs are found in bone marrow and spleen at late stage of leukemia. - Abstract: Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca{sup 2+} response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

  14. 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

  15. Tachykinin receptors mediating airway marcomolecular secretion

    SciTech Connect

    Gentry, S.E. )

    1991-01-01

    Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with {sup 3}H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers.

  16. 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

  17. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

    PubMed Central

    Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

    2015-01-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

  18. Purinergic Receptors in Thrombosis and Inflammation.

    PubMed

    Hechler, Béatrice; Gachet, Christian

    2015-11-01

    Under various pathological conditions, including thrombosis and inflammation, extracellular nucleotide levels may increase because of both active release and passive leakage from damaged or dying cells. Once in the extracellular compartment, nucleotides interact with plasma membrane receptors belonging to the P2 purinergic family, which are expressed by virtually all circulating blood cells and in most blood vessels. In this review, we focus on the specific role of the 3 platelet P2 receptors P2Y1, P2Y12, and P2X1 in hemostasis and arterial thrombosis. Beyond platelets, these 3 receptors, along with the P2Y2, P2Y6, and P2X7 receptors, constitute the main P2 receptors mediating the proinflammatory effects of nucleotides, which play important roles in various functions of circulating blood cells and cells of the vessel wall. Each of these P2 receptor subtypes specifically contributes to chronic or acute vascular inflammation and related diseases, such as atherosclerosis, restenosis, endotoxemia, and sepsis. The potential for therapeutic targeting of these P2 receptor subtypes is also discussed.

  19. 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.

  20. Principles of antibody-mediated TNF receptor activation

    PubMed Central

    Wajant, H

    2015-01-01

    From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

  1. 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.

  2. 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.

  3. 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.

  4. Potential Involvement of P2 Receptors in the Pathological Processes of Hyperthyroidism: A Pilot Study.

    PubMed

    Hong, Wu; Li, Guodong; Nie, Yijun; Zou, Lifang; Zhang, Xi; Liu, Shuangmei; Li, Guilin; Xu, Hong; Zhang, Chun-Ping; Liang, Shangdong

    2016-05-01

    Symptoms of hyperthyroidism manifest mainly as changes in the nervous and metabolic systems. Whether P2X receptors (ionotropic ATP purinergic receptors, including P2X3 receptor and P2X7 receptor) are involved in the alterations of these disorders still remains unclear. Thus, this study aimed to assess the association of hyperthyroidism with the expression of P2X3 and P2X7 receptors and the concentrations of ATP in blood leukocytes and catecholamine. Twelve healthy subjects and twelve patients diagnosed with hyperthyroidism were recruited. Serum free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) levels had been detected by chemiluminescence method. Meanwhile, the catecholamine levels (including adrenaline, noradrenaline, and dopamine) in plasma, ATP level and P2X receptors (including P2X3 receptor and P2X7 receptor) in peripheral blood had been detected by high performance liquid chromatography, bioluminescence method, and reverse transcription polymerase chain reaction, respectively. Levels of epinephrine and norepinephrine were significantly higher in the hyperthyroidism group compared with the control group. The concentration of ATP in the hyperthyroidism group was significantly higher than its in the control group. The expression of P2X3 mRNA and P2X7 mRNA in hyperthyroidism group were significantly increased compared with those in control group. In a conclusion, there is a relationship between the elevated expression of P2X3 receptor and P2X7 receptor in peripheral blood leukocytes and high serum epinephrine and norepinephrine levels in hyperthyroidism patients. PMID:27312548

  5. Resveratrol inhibits glycine receptor-mediated ion currents.

    PubMed

    Lee, Byung-Hwan; Hwang, Sung-Hee; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Lee, Joon-Hee; Kim, Hyung-Chun; Rhim, Hyewhon; Nah, Seung-Yeol

    2014-01-01

    Resveratrol is found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-nociceptive, and life-prolonging effects. However, the single cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. The glycine receptor is an inhibitory ligand-gated ion channel involved in fast synaptic transmission in spinal cord. In the present study, we investigated the effect of resveratrol on human glycine receptor channel activity. Glycine α1 receptors were expressed in Xenopus oocytes and glycine receptor channel activity was measured using a two-electrode voltage clamp technique. Treatment with resveratrol alone had no effect on oocytes injected with H2O or on oocytes injected with glycine α1 receptor cRNA. In the oocytes injected with glycine α1 receptor cRNA, co- or pre-treatment of resveratrol with glycine inhibited the glycine-induced inward peak current (IGly) in a reversible manner. The inhibitory effect of resveratrol on IGly was also concentration dependent, voltage independent, and non-competitive. These results indicate that resveratrol regulates glycine receptor channel activity and that resveratrol-mediated regulation of glycine receptor channel activity is one of several cellular action mechanisms of resveratrol for pain regulation. PMID:24694604

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

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

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

  7. 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.

  8. GABAA receptor-mediated neurotransmission: Not so simple after all.

    PubMed

    Knoflach, Frédéric; Hernandez, Maria-Clemencia; Bertrand, Daniel

    2016-09-01

    GABAA receptors are ligand-gated ion channels that form a fundamental component of inhibitory neurotransmission in the central and peripheral nervous systems. However, since the initial recordings of inhibitory electrical activity of neurons in response to GABA, these receptors have been found to play a more complex role and can, under some circumstances, function in an excitatory manner. This has been demonstrated via electrophysiological recordings conducted in both mature and developing neurons from different brain regions, as well as in various subcellular locations such as dendritic and axonal membranes. The balance between the inhibitory and excitatory effects mediated by GABAA receptor activation depends not only on multiple factors that govern the equilibrium of the transmembrane chloride gradient, but also on bicarbonate concentration. Moreover, electrophysiological and fluorescence measurements have revealed that a spatial distribution of the chloride gradient exists within neurons, which locally influences the effects mediated by GABAA receptor activation. In recent years, it has also become apparent that intra-neuronal chloride concentration is partially regulated by cation-chloride co-transporters (CCCs), in particular NKCC1 and KCC2. The aim of the present commentary is to discuss, in light of the latest findings, potential implications of the tight spatial and temporal regulation of chloride equilibrium in health and disease, as well as its relevance for the therapeutic effects of molecules acting at GABAA receptors. PMID:27002180

  9. Pharmacology of inflammatory pain: local alteration in receptors and mediators

    PubMed Central

    Holzer, Peter; Holzer-Petsche, Ulrike

    2015-01-01

    Background Inflammation is commonly associated with hyperalgesia. Ideally, this change should abate once inflammation is resolved but this is not necessarily the case because phenotypic changes in the tissue can persist, as appears to be the case in postinfectious irritable bowel syndrome. Basically, all primary afferent neurons supplying the gut can sensitize in response to proinflammatory mediators, and the mechanisms whereby hypersensitivity is initiated and maintained are thus of prime therapeutic interest. Experimental and clinical findings There is a multitude of molecular nocisensors that can be responsible for the hypersensitivity of afferent neurons. These entities include: (i) receptors and sensors at the peripheral terminals of afferent neurons that are relevant to stimulus transduction, (ii) ion channels that govern the excitability and conduction properties of afferent neurons, and (iii) transmitters and transmitter receptors that mediate communication between primary afferents and second-order neurons in the spinal cord and brainstem. Persistent increases in the sensory gain may result from changes in the expression of transmitters, receptors and ion channels, changes in the subunit composition and biophysical properties of receptors and ion channels or changes in the structure, connectivity and survival of afferent neurons. Particular therapeutic potential is attributed to targets that are selectively expressed by afferent neurons and whose number and function are altered in abdominal hypersensitivity. Conclusion Emerging targets of therapeutic relevance include distinct members of the transient receptor potential (TRP) channel family (TRPV1, TRPV4, TRPA1), acid-sensing ion channels, protease-activated receptors, corticotropin-releasing factor receptors and sensory neuron-specific sodium channels. PMID:20203494

  10. 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

  11. 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.

  12. Purinergic Receptors: Key Mediators of HIV-1 Infection and Inflammation

    PubMed Central

    Swartz, Talia H.; Dubyak, George R.; Chen, Benjamin K.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) causes a chronic infection that afflicts more than 30 million individuals worldwide. While the infection can be suppressed with potent antiretroviral therapies, individuals infected with HIV-1 have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV-1 pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here, we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets. PMID:26635799

  13. 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.

  14. Of pheromones and kairomones: what receptors mediate innate emotional responses?

    PubMed

    Fortes-Marco, Lluis; Lanuza, Enrique; Martinez-Garcia, Fernando

    2013-09-01

    Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation.

  15. Virus-receptor interactions and receptor-mediated virus entry into host cells.

    PubMed

    Casasnovas, José M

    2013-01-01

    The virus particles described in previous chapters are vehicles that transmit the viral genome and the infection from cell to cell. To initiate the infective cycle, the viral genome must therefore translocate from the viral particle to the cytoplasm. Via distinct proteins or motifs in their outermost shell, the particles attach initially to specific molecules on the host cell surface. These virus receptors thus mediate penetration of the viral genome inside the cell, where the intracellular infective cycle starts. The presence of these receptors on the cell surface is a principal determinant of virus host tropism. Viruses can use diverse types of molecules to attach to and enter into cells. In addition, virus-receptor recognition can evolve over the course of an infection, and virus variants with distinct receptor-binding specificities and tropism can appear. The identification of virus receptors and the characterization of virus-receptor interactions have been major research goals in virology for the last two decades. In this chapter, we will describe, from a structural perspective, several virus-receptor interactions and the active role of receptor molecules in virus entry. PMID:23737061

  16. TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation

    PubMed Central

    Kunkler, Phillip Edward; Ballard, Carrie Jo; Oxford, Gerry Stephen; Hurley, Joyce Harts

    2010-01-01

    The TRPA1 receptor is a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons. TRPA1 receptors are targeted by pungent compounds from mustard and garlic and environmental irritants such as formaldehyde and acrolein. Ingestion or inhalation of these chemical agents causes irritation and burning in the nasal and oral mucosa and respiratory lining. Headaches have been widely reported to be induced by inhalation of environmental irritants, but it is unclear how these agents produce headache. Stimulation of trigeminal neurons releases CGRP and substance P and induces neurogenic inflammation associated with the pain of migraine. Here we test the hypothesis that activation of TRPA1 receptors are the mechanistic link between environmental irritants and peptide mediated neurogenic inflammation. Known TRPA1 agonists and environmental irritants stimulate CGRP release from dissociated rat trigeminal ganglia neurons and this release is blocked by a selective TRPA1 antagonist, HC-030031. Further, TRPA1 agonists and environmental irritants increase meningeal blood flow following intranasal administration. Prior dural application of the CGRP antagonist, CGRP8–37, or intranasal or dural administration of HC-030031, blocks the increases in blood flow elicited by environmental irritants. Together these results demonstrate that TRPA1 receptor activation by environmental irritants stimulates CGRP release and increases cerebral blood flow. We suggest that these events contribute to headache associated with environmental irritants. PMID:21075522

  17. 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

  18. Formylpeptide receptors mediate rapid neutrophil mobilization to accelerate wound healing.

    PubMed

    Liu, Mingyong; Chen, Keqiang; Yoshimura, Teizo; Liu, Ying; Gong, Wanghua; Le, Yingying; Gao, Ji-Liang; Zhao, Jianhua; Wang, Ji Ming; Wang, Aimin

    2014-01-01

    Wound healing is a multi-phased pathophysiological process requiring chemoattractant receptor-dependent accumulation of myeloid cells in the lesion. Two G protein-coupled formylpeptide receptors Fpr1 and Fpr2 mediate rapid neutrophil infiltration in the liver of Listeria-infected mice by sensing pathogen-derived chemotactic ligands. These receptors also recognize host-derived chemotactic peptides in inflammation and injury. Here we report the capacity of Fprs to promote the healing of sterile skin wound in mice by initiating neutrophil infiltration. We found that in normal miceneutrophils rapidly infiltrated the dermis in the wound before the production of neutrophil-specific chemokines by the injured tissue. In contrast, rapid neutrophil infiltration was markedly reduced with delayed wound closure in mice deficient in both Fprs. In addition, we detected Fpr ligand activity that chemoattracted neutrophils into the wound tissue. Our study thus demonstrates that Fprs are critical for normal healing of the sterile skin wound by mediating the first wave of neutrophil infiltration.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  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. The mechanisms of HAMP-mediated signaling in transmembrane receptors.

    PubMed

    Ferris, Hedda U; Dunin-Horkawicz, Stanislaw; Mondéjar, Laura García; Hulko, Michael; Hantke, Klaus; Martin, Jörg; Schultz, Joachim E; Zeth, Kornelius; Lupas, Andrei N; Coles, Murray

    2011-03-01

    HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.

  6. VEGF receptors mediate hypoxic remodeling of adult ovine carotid arteries.

    PubMed

    Adeoye, Olayemi O; Bouthors, Vincent; Hubbell, Margaret C; Williams, James M; Pearce, William J

    2014-10-01

    Recent studies suggest that VEGF contributes to hypoxic remodeling of arterial smooth muscle, although hypoxia produces only transient increases in VEGF that return to normoxic levels despite sustained changes in arterial structure and function. To explore how VEGF might contribute to long-term hypoxic vascular remodeling, this study explores the hypothesis that chronic hypoxia produces sustained increases in smooth muscle VEGF receptor density that mediate long-term vascular effects of hypoxia. Carotid arteries from adult sheep maintained at sea level or altitude (3,820 m) for 110 days were harvested and denuded of endothelium. VEGF levels were similar in chronically hypoxic and normoxic arteries, as determined by immunoblotting. In contrast, VEGF receptor levels were significantly increased by 107% (VEGF-R1) and 156% (VEGF-R2) in hypoxic compared with normoxic arteries. In arteries that were organ cultured 24 h with 3 nM VEGF, VEGF replicated effects of hypoxia on abundances of smooth muscle α actin (SMαA), myosin light chain kinase (MLCK), and MLC20 and the effects of hypoxia on colocalization of MLC20 with SMαA, as measured via confocal microscopy. VEGF did not replicate the effects of chronic hypoxia on colocalization of MLCK with SMαA or MLCK with MLC20, suggesting that VEGF's role in hypoxic remodeling is highly protein specific, particularly for contractile protein organization. VEGF effects in organ culture were inhibited by VEGF receptor blockers vatalinib (240 nM) and dasatinib (6.3 nM). These findings support the hypothesis that long-term upregulation of VEGF receptors help mediate sustained effects of hypoxia on the abundance and colocalization of contractile proteins in arterial smooth muscle. PMID:25038104

  7. Crosstalk between purinergic receptors and lipid mediators in leishmaniasis.

    PubMed

    Chaves, Mariana M; Canetti, Cláudio; Coutinho-Silva, Robson

    2016-01-01

    Leishmaniasis is a neglected tropical disease affecting millions of people around the world caused by organisms of the genus Leishmania. Parasite escape mechanisms of the immune system confer the possibility of resistance and dissemination of the disease. A group of molecules that has become a target for Leishmania survival strategies are lipid mediators. Among them, leukotriene B4 (LTB4) has been described as a pro-inflammatory molecule capable of activating cells of the immune system to combat Leishmania. In an opposite way, prostaglandin E2 (PGE2) is a lipid mediator described as a deactivator of macrophages and neutrophils. The balance of these two molecules can be generated by extracellular nucleotides, such as adenosine 5'-triphosphate (ATP) and adenosine (Ado), which activate the purinergic receptors system. Herein, we discuss the role of extracellular nucleotides and the resulting balance of LTB4 and PGE2 in Leishmania fate, survival or death. PMID:27595742

  8. 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.

  9. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines

    PubMed Central

    Üçpunar, Habibe K.; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C.

    2016-01-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly’s high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans. PMID:27145030

  10. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

    PubMed

    Hussain, Ashiq; Zhang, Mo; Üçpunar, Habibe K; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C

    2016-05-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly's high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans. PMID:27145030

  11. 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

  12. The type I interleukin-1 receptor mediates fever in the rat as shown by interleukin-1 receptor subtype selective ligands.

    PubMed

    Malinowsky, D; Chai, Z; Bristulf, J; Simoncsits, A; Bartfai, T

    1995-12-01

    The interleukin-1 (IL-1) system possesses two distinct receptors (type I and type II) which, together with the accessory protein, mediate a multitude of responses to IL-1 alpha and IL-1 beta, including fever. So far, no receptor subtype-specific ligands have been described. Since both types of IL-1 receptors occur in the thermoregulatory areas it was unclear which IL-1 receptor type mediates fever. We report here that for a series of deletion mutants of human recombinant IL-1 beta (hrIL-1 beta), the affinity of these ligands for the type I IL-1 receptor correlates with their efficacy to evoke the fever response (hrIL-1 beta > des-SND52-54 > des-QGE48-50 > des-I56). Thus, the results suggest that agonist occupancy of the type I IL-1 receptor is essential for IL-1 beta-mediated fever.

  13. 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

  14. 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

  15. 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.

  16. Hepatoprotective actions of melatonin: Possible mediation by melatonin receptors

    PubMed Central

    Mathes, Alexander M

    2010-01-01

    Melatonin, the hormone of darkness and messenger of the photoperiod, is also well known to exhibit strong direct and indirect antioxidant properties. Melatonin has previously been demonstrated to be a powerful organ protective substance in numerous models of injury; these beneficial effects have been attributed to the hormone’s intense radical scavenging capacity. The present report reviews the hepatoprotective potential of the pineal hormone in various models of oxidative stress in vivo, and summarizes the extensive literature showing that melatonin may be a suitable experimental substance to reduce liver damage after sepsis, hemorrhagic shock, ischemia/reperfusion, and in numerous models of toxic liver injury. Melatonin’s influence on hepatic antioxidant enzymes and other potentially relevant pathways, such as nitric oxide signaling, hepatic cytokine and heat shock protein expression, are evaluated. Based on recent literature demonstrating the functional relevance of melatonin receptor activation for hepatic organ protection, this article finally suggests that melatonin receptors could mediate the hepatoprotective actions of melatonin therapy. PMID:21182223

  17. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

    PubMed Central

    Xing, Shu; Grol, Matthew W.; Grutter, Peter H.; Dixon, S. Jeffrey; Komarova, Svetlana V.

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP. PMID:27468270

  18. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    PubMed

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP. PMID:27468270

  19. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    PubMed

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  20. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    EPA Science Inventory

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
    Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  1. 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

  2. Identification and Expression Analysis of Putative Chemosensory Receptor Genes in Microplitis mediator by Antennal Transcriptome Screening

    PubMed Central

    Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Gu, Shao-Hua; Li, Rui-Jun; Zhou, Jing-Jiang; Zhang, Yong-Jun; Guo, Yu-Yuan

    2015-01-01

    Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor perception through sensory organs such as antennae. Antennal chemoreception is extremely important for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level. PMID:26078716

  3. 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

  4. CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

    PubMed Central

    Miller, Rachel E.; Tran, Phuong B.; Das, Rosalina; Ghoreishi-Haack, Nayereh; Ren, Dongjun; Miller, Richard J.; Malfait, Anne-Marie

    2012-01-01

    Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain. PMID:23185004

  5. Presynaptic Kainate Receptor Mediation of Frequency Facilitation at Hippocampal Mossy Fiber Synapses

    NASA Astrophysics Data System (ADS)

    Schmitz, Dietmar; Mellor, Jack; Nicoll, Roger A.

    2001-03-01

    Inhibition of transmitter release by presynaptic receptors is widespread in the central nervous system and is typically mediated via metabotropic receptors. In contrast, very little is known about facilitatory receptors, and synaptic activation of a facilitatory autoreceptor has not been established. Here we show that activation of presynaptic kainate receptors can facilitate transmitter release from hippocampal mossy fiber synapses. Synaptic activation of these presumed ionotropic kainate receptors is very fast (<10 ms) and lasts for seconds. Thus, these presynaptic kainate receptors contribute to the short-term plasticity characteristics of mossy fiber synapses, which were previously thought to be an intrinsic property of the synapse.

  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. A novel member of the integrin receptor family mediates Arg-Gly-Asp- stimulated neutrophil phagocytosis

    PubMed Central

    1989-01-01

    Human neutrophils (PMN) express a heterodimeric receptor that has ligand binding specificity for the Arg-Gly-Asp (RGD) sequence within many adhesive proteins. A monoclonal antibody, B6H12, which binds to this receptor, inhibits both RGD-mediated ligand binding and stimulation of IgG-mediated phagocytosis by fibronectin, fibrinogen, vitronectin, von Willebrand's factor, and collagen type IV. By several criteria this receptor is neither a known very late antigen, a known cytoadhesin (gp IIb/IIIa-vitronectin receptor), nor a member of the LFA- 1, Mac-1, p150,95 group of integrin receptors. Ligand binding via this receptor is rapidly inactivated by products of the myeloperoxidase- hydrogen peroxide-halide system of PMN. We conclude that this receptor, for which we propose the name leukocyte response integrin, is a signal- transducing molecule on PMN which may have a significant early role in modulation of PMN function at inflammatory sites. PMID:2785522

  8. 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

  9. 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

  10. Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases.

    PubMed

    Mbengue, Malick; Bourdais, Gildas; Gervasi, Fabio; Beck, Martina; Zhou, Ji; Spallek, Thomas; Bartels, Sebastian; Boller, Thomas; Ueda, Takashi; Kuhn, Hannah; Robatzek, Silke

    2016-09-27

    Sensing of potential pathogenic bacteria is of critical importance for immunity. In plants, this involves plasma membrane-resident pattern recognition receptors, one of which is the FLAGELLIN SENSING 2 (FLS2) receptor kinase. Ligand-activated FLS2 receptors are internalized into endosomes. However, the extent to which these spatiotemporal dynamics are generally present among pattern recognition receptors (PRRs) and their regulation remain elusive. Using live-cell imaging, we show that at least three other receptor kinases associated with plant immunity, PEP RECEPTOR 1/2 (PEPR1/2) and EF-TU RECEPTOR (EFR), internalize in a ligand-specific manner. In all cases, endocytosis requires the coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1), and thus depends on receptor activation status. We also show the internalization of liganded FLS2, suggesting the transport of signaling competent receptors. Trafficking of activated PRRs requires clathrin and converges onto the same endosomal vesicles that are also shared with the hormone receptor BRASSINOSTERIOD INSENSITIVE 1 (BRI1). Importantly, clathrin-dependent endocytosis participates in plant defense against bacterial infection involving FLS2-mediated stomatal closure and callose deposition, but is uncoupled from activation of the flagellin-induced oxidative burst and MAP kinase signaling. In conclusion, immunity mediated by pattern recognition receptors depends on clathrin, a critical component for the endocytosis of signaling competent receptors into a common endosomal pathway. PMID:27651493

  11. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    PubMed

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction.

  12. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  13. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking

    ERIC Educational Resources Information Center

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  14. Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells

    PubMed Central

    Pang, Ji-Jie; Gao, Fan; Wu, Samuel M.

    2013-01-01

    Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons. PMID:22842089

  15. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns (PAMPs) and endogenous molecules...

  16. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  17. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor.

    PubMed

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas

    2015-12-01

    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  18. Glutamate mediates the function of melanocortin receptor 4 on sim1 neurons in body weight regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing ...

  19. Steroid receptor coactivator-1 mediates estrogenic actions to prevent body weight gain in female mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estrogen receptor-alpha (ERalpha) expressed by hypothalamic proopiomelanocortin and steroidogenic factor-1 neurons largely mediates the antiobesity effects of estrogens in females. However, the critical molecular events that are coupled to ERalpha and mediate estrogenic effects on energy balance rem...

  20. 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.

  1. Activation of the Tonic GABAC Receptor Current in Retinal Bipolar Cell Terminals by Nonvesicular GABA Release

    PubMed Central

    Jones, S. M.; Palmer, M. J.

    2009-01-01

    Within the second synaptic layer of the retina, bipolar cell (BC) output to ganglion cells is regulated by inhibitory input to BC axon terminals. GABAA receptors (GABAARs) mediate rapid synaptic currents in BC terminals, whereas GABAC receptors (GABACRs) mediate slow evoked currents and a tonic current, which is strongly regulated by GAT-1 GABA transporters. We have used voltage-clamp recordings from BC terminals in goldfish retinal slices to determine the source of GABA for activation of these currents. Inhibition of vesicular release with concanamycin A or tetanus toxin significantly inhibited GABAAR inhibitory postsynaptic currents and glutamate-evoked GABAAR and GABACR currents but did not reduce the tonic GABACR current, which was also not dependent on extracellular Ca2+. The tonic current was strongly potentiated by inhibition of GABA transaminase, under both normal and Ca2+-free conditions, and was activated by exogenous taurine; however inhibition of taurine transport had little effect. The tonic current was unaffected by GAT-2/3 inhibition and was potentiated by GAT-1 inhibition even in the absence of vesicular release, indicating that it is unlikely to be evoked by reversal of GABA transporters or by ambient GABA. In addition, GABA release does not appear to occur via hemichannels or P2X7 receptors. BC terminals therefore exhibit two forms of GABACR-mediated inhibition, activated by vesicular and by nonvesicular GABA release, which are likely to have distinct functions in visual signal processing. The tonic GABACR current in BC terminals exhibits similar properties to tonic GABAAR and glutamate receptor currents in the brain. PMID:19494193

  2. 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

  3. 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

    Benzene can induce hematotoxicity and leukemia in humans and mice. Since a review of the literature shows that the CYP2E1 knockout mouse is not known to possess any benzene toxicity, the metabolism of benzene by CYP2E1 in the liver is regarded to be prerequisite for its cytotoxicity and genotoxicity, although the mechanism is not fully understood yet. Because it was found some years ago that benzene was also a substrate for CYP1A1, we investigated the involvement of the aryl hydrocarbon receptor (AhR) in benzene hematotoxicity using AhR wild-type (AhR(+/+)), heterozygous (AhR(+/-)), and homozygous (AhR(-/-)) male mice. Interestingly, following a 2-week inhalation of 300 ppm benzene (a potent dose for leukemogenicity), no hematotoxicity was induced in AhR(-/-) mice. Further, there were no changes in cellularity of peripheral blood and bone marrow (BM), nor in levels of granulocyte-macrophage colony-forming units in BM. This lack of hematotoxicity was associated with the lack of p21 overexpression, which was regularly seen in the wild-type mice following benzene inhalation. Combined treatment with two major benzene metabolites, phenol and hydroquinone, induced hemopoietic toxicity, although it was not known whether this happened due to a surprising lack of expression of CYP2E1 by AhR knockout, or due to a lack of other AhR-mediated CYP enzymes, including 1A1 (i.e., a possible alternative pathway of benzene metabolism). The former possibility, evaluated in the present study, failed to show a significant relationship between AhR and the expression of CYP2E1. Furthermore, a subsequent evaluation of AhR expression after benzene inhalation tended to show higher but less significant expression in the liver, and none in the BM, compared with sham control. Although this study failed to identify the more likely of the above-mentioned two possibilities, the study using AhR knockout mice on benzene inhalation presents the unique possibility that the benzene toxicity may be

  4. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

    PubMed Central

    Delgado-Peraza, Francheska; Ahn, Kwang H.; Nogueras-Ortiz, Carlos; Mungrue, Imran N.; Mackie, Ken; Kendall, Debra A.

    2016-01-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  5. 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

  6. 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

  7. Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake

    PubMed Central

    Rodríguez-Vázquez, Míriam; Mejía-Morales, John E.; Culi, Joaquim

    2015-01-01

    Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP). We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them. PMID:26121667

  8. Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain

    PubMed Central

    Viatchenko-Karpinski, Viacheslav; Novosolova, Natalia; Ishchenko, Yevheniia; Azhar, M Ameruddin; Wright, Michael; Tsintsadze, Vera; Kamal, Ahmed; Burnashev, Nail; Voitenko, Nana; Giniatullin, Rashid; Lozovaya, Natalia

    2016-01-01

    Background A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons. Results The effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons). Thereafter, the action of these stable, synthetic Ap4A analogs on inflammatory pain and thermal hyperalgesia is studied through the measurement of antinociceptive effects in formalin and Hargreaves plantar tests in rats in vivo. In vitro inhibition of rat P2X3Rs (not P2X2Rs, P2X4Rs nor P2X7Rs) is shown to take place mediated by high-affinity desensitization (at low concentrations; IC50 values 100–250 nM) giving way to only weak partial agonism at much higher concentrations (EC50 values ≥ 10 µM). Similar inhibitory activity is observed with human recombinant P2X3Rs. The inhibitory effects of AppNHppA on nodose, dorsal root, and trigeminal neuron whole cell currents suggest that stable, synthetic Ap4A analogs inhibit homomeric P2X3Rs in preference to heteromeric P2X2/3Rs. Both Ap4A analogs mediate clear inhibition of pain responses in both in vivo inflammation models. Conclusions Stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) being weak partial agonist provoke potent high-affinity desensitization-mediated inhibition of homomeric P2X3Rs at low concentrations. Therefore, both analogs demonstrate clear potential as potent analgesic agents for use in the management of chronic pain associated with heightened P2X3R activation. PMID:27030723

  9. TNF-Α May Mediate Inflammasome Activation in the Absence of Bacterial Infection in More than One Way

    PubMed Central

    Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2013-01-01

    Members of the mammalian nucleotide binding domain, leucine-rich repeat (LRR)-containing receptor family of proteins are key modulators of innate immunity regulating inflammation. To date, microbial pathogen-associated molecules and toxins have been identified as key triggers of activation of inflammasomes. However, recently, environmental, and neurodegenerative stimuli have been identified that lead to IL-1β release by means of inflammasomes. IL-1β plays a crucial role during brain inflammation, and caspase-1 appears to be a key modulator of IL-1β bioactivity and the consequent transcriptional regulation of gene expression within the brain during inflammation. We show here that exposure of a human neuroblastoma cell line (SK-N-MC cells) to TNF-α promotes ROS-mediated caspase-1 activation and IL-1β secretion. The involvement of NF-κB in the regulation of IL-1β synthesis is investigated through specific inhibition of this transcription factor. The effect of TNF-α was abolished in the presence of ROS inhibitors as NAC, or DPI. Remarkably, SK-N-MC cells do not respond to ATP stimulation in spite of P2X7R expression. These results provide a mechanism by which danger signals and particulate matter mediate inflammation via the inflammasome in the absence of microbial infection. PMID:23940760

  10. P2×7 Receptor in the Kidneys of Diabetic Rats Submitted to Aerobic Training or to N-Acetylcysteine Supplementation

    PubMed Central

    Rodrigues, Adelson M.; Bergamaschi, Cassia T.; Fernandes, Maria Jose S.; Paredes-Gamero, Edgar J.; Curi, Marcus V.; Ferreira, Alice T.; Araujo, Sergio R. R.; Punaro, Giovana R.; Maciel, Fabiane R.; Nogueira, Guilherme B.; Higa, Elisa M. S.

    2014-01-01

    Previous studies in our laboratory showed that N-acetylcysteine supplementation or aerobic training reduced oxidative stress and the progression of diabetic nephropathy in rats. The P2X7 receptor is up-regulated in pathological conditions, such as diabetes mellitus. This up-regulation is related to oxidative stress and induces tissue apoptosis or necrosis. The aim of the present study is to assess the role of P2X7 receptor in the kidneys of diabetic rats submitted to aerobic training or N-acetylcysteine supplementation. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, i.v.) and the training was done on a treadmill; N-acetylcysteine was given in the drinking water (600 mg/L). By confocal microscopy, as compared to control, the kidneys of diabetic rats showed increased P2×7 receptor expression and a higher activation in response to 2′(3′)-O-(4-benzoylbenzoyl) adenosine5'–triphosphate (specific agonist) and adenosine triphosphate (nonspecific agonist) (all p<0.05). All these alterations were reduced in diabetic rats treated with N-acetylcysteine, exercise or both. We also observed measured proteinuria and albuminuria (early marker of diabetic nephropathy) in DM groups. Lipoperoxidation was strongly correlated with P2X7 receptor expression, which was also correlated to NO•, thus associating this receptor to oxidative stress and kidney lesion. We suggest that P2X7 receptor inhibition associated with the maintenance of redox homeostasis could be useful as coadjuvant treatment to delay the progression of diabetic nephropathy. PMID:24940871

  11. Caspase-1-mediated cytokine release from gestational tissues, placental, and cord blood

    PubMed Central

    Maneta, Ebtehaj; Warren, Averil Y.; Hay, Daniel P.; Khan, Raheela N.

    2015-01-01

    Distinguishing between fetal and maternal inflammatory responses is necessary for understanding the immune interplay either side of the placenta. Fetal immunity reaches maturity during extrauterine life and while basic inflammatory responses afford a certain degree of protection, fetuses are vulnerable to infection. With the discovery of inflammasomes—intracellular scaffolds that facilitate the elaboration of reactions resulting in the release of mature interleukin-1β (IL-1β)—it is necessary to consider how inflammatory stimuli are processed. The purinergic P2X7 receptor located on haematopoietic cells is a key intermediary in signal transduction initiated at Toll-like receptors (TLR) terminating in release of the mature IL-1β product. We demonstrate herein that IL-1β release from fetal membranes and mononuclear cells isolated from cord, placental, and maternal blood, obtained at term, is P2X7- and caspase-1 dependent. The P2X7-dependent release of the cytokine, which was highest from choriodecidua, was attenuated by progesterone (P4), prolactin and an NFkB inhibitor. The NLRP3 inflammasome appears necessary for the processing of IL-1β in gestational tissues and leukocytes. PMID:26157394

  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. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  14. EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury

    PubMed Central

    Tsenkina, Y; Ricard, J; Runko, E; Quiala- Acosta, M M; Mier, J; Liebl, D J

    2015-01-01

    We demonstrate that EphB3 receptors mediate oligodendrocyte (OL) cell death in the injured spinal cord through dependence receptor mechanism. OLs in the adult spinal cord express EphB3 as well as other members of the Eph receptor family. Spinal cord injury (SCI) is associated with tissue damage, cellular loss and disturbances in EphB3-ephrinB3 protein balance acutely (days) after the initial impact creating an environment for a dependence receptor-mediated cell death to occur. Genetic ablation of EphB3 promotes OL survival associated with increased expression of myelin basic protein and improved locomotor function in mice after SCI. Moreover, administration of its ephrinB3 ligand to the spinal cord after injury also promotes OL survival. Our in vivo findings are supported by in vitro studies showing that ephrinB3 administration promotes the survival of both oligodendroglial progenitor cells and mature OLs cultured under pro-apoptotic conditions. In conclusion, the present study demonstrates a novel dependence receptor role of EphB3 in OL cell death after SCI, and supports further development of ephrinB3-based therapies to promote recovery. PMID:26469970

  15. Benzodiazepine receptor ligand influences on acquisition: suggestion of an endogenous modulatory mechanism mediated by benzodiazepine receptors.

    PubMed

    Izquierdo, I; Pereira, M E; Medina, J H

    1990-07-01

    In rats, pretraining ip administration of the central benzodiazepine receptor antagonist, flumazenil (5.0 mg/kg), or of the inverse agonist, n-butyl-beta-carboline-3-carboxylate (BCCB) (0.2 or 0.5 mg/kg), facilitated retention of a step-down inhibitory avoidance task; the central agonists, clonazepam and diazepam (0.4 or 1.0 mg/kg), had an opposite effect, and the peripheral agonist, 4'-chlordiazepam (1.25 or 6.25 mg/kg), was without effect. Pre- but not post-training flumazenil (2.0 mg/kg) blocked the effect of BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), or diazepam (1.0 mg/kg) given also pretraining. The post-training administration of all of these drugs had no effect on retention of the avoidance task. Flumazenil (5.0 mg/kg) and BCCB (0.5 mg/kg), given before training, enhanced retention test performance of habituation to a buzzer but not of habituation to an open field. In the three tasks studied, none of the drugs used had any appreciable effect on training session parameters. These results suggest that there is an endogenous mechanism mediated by benzodiazepine agonists, sensitive to inverse agonists, that normally down-regulates acquisition of certain behaviors; this mechanism becomes activated only when the tasks involve or occur with a certain degree of stress or anxiety (i.e., inhibitory avoidance or habituation to the buzzer) and not in less stressful or anxiogenic tasks (i.e., habituation to an open field).

  16. The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila

    PubMed Central

    Ni, Lina; Klein, Mason; Svec, Kathryn V; Budelli, Gonzalo; Chang, Elaine C; Ferrer, Anggie J; Benton, Richard; Samuel, Aravinthan DT; Garrity, Paul A

    2016-01-01

    Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity. DOI: http://dx.doi.org/10.7554/eLife.13254.001 PMID:27126188

  17. G-1-activated membrane estrogen receptors mediate increased contractility of the human myometrium.

    PubMed

    Maiti, K; Paul, J W; Read, M; Chan, E C; Riley, S C; Nahar, P; Smith, R

    2011-06-01

    Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity. PMID:21427217

  18. Corticotropin-releasing factor CRF1, but not CRF2, receptors mediate anxiogenic-like behavior.

    PubMed

    Heinrichs, S C; Lapsansky, J; Lovenberg, T W; De Souza, E B; Chalmers, D T

    1997-07-23

    The recent identification and differential localization in brain of three binding sites for corticotropin-releasing factor (CRF)-like peptides (CRF1 and CRF2 receptors as well as CRF-binding protein) suggest the existence of functionally distinct neurobiological systems which mediate CRF activation. For instance, evidence from receptor knockdown and pharmacological studies suggest involvement of the CRF1 receptor in anxiogenic-like behavior and the CRF-binding protein in learning and memory processes. The present studies examined the potential functional significance of the CRF2 receptor in relation to the CRF1 receptor using two animal models of anxiety and endocrine reactivity to a stressor. CRF1 and CRF2 receptor knockdown was achieved and confirmed autoradiographically within brain regions relevant to behavioral reactivity to stressors by chronic, central administration of antisense oligonucleotides. CRF1 but not CRF2, know down produced a significant anxiolytic-like effect in the Defensive Withdrawal relative to vehicle-treated and two missense oligonucleotide negative control groups. In contrast, neither antisense treatment altered endocrine or behavioral reactivity to a swim stressor. Thus, the present data support the reported role of CRF1 receptors in the mediation of anxiogenic-like behavior and suggest a functionally distinct for role for CRF2 receptors in brain.

  19. 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.

  20. 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.

  1. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons.

    PubMed

    Corey, Elizabeth A; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W

    2010-04-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

  2. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  3. 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

  4. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  5. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

    PubMed Central

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

  6. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  7. κ-Opioid receptors within the nucleus accumbens shell mediate pair bond maintenance.

    PubMed

    Resendez, Shanna L; Kuhnmuench, Morgan; Krzywosinski, Tarin; Aragona, Brandon J

    2012-05-16

    The prairie vole is a socially monogamous species in which breeder pairs typically show strong and selective pair bonds. The establishment of a pair bond is associated with a behavioral transition from general affiliation to aggressive rejection of novel conspecifics. This "selective aggression" is indicative of mate guarding that is necessary to maintain the initial pair bond. In the laboratory, the neurobiology of this behavior is studied using resident-intruder testing. Although it is well established that social behaviors in other species are mediated by endogenous opioid systems, opiate regulation of pair bond maintenance has never been studied. Here, we used resident-intruder testing to determine whether endogenous opioids within brain motivational circuitry mediate selective aggression in prairie voles. We first show that peripheral blockade of κ-opioid receptors with the antagonist norbinaltorphimine (nor-BNI; 100 mg/kg), but not with the preferential μ-opioid receptor antagonist naloxone (1, 10, or 30 mg/kg), decreased selective aggression in males. We then provide the first comprehensive characterization of κ- and μ-opioid receptors in the prairie vole brain. Finally, we demonstrate that blockade of κ-opioid receptors (500 ng nor-BNI) within the nucleus accumbens (NAc) shell abolishes selective aggression in both sexes, but blockade of these receptors within the NAc core enhances this behavior specifically in females. Blockade of κ-opioid receptors within the ventral pallidum or μ-opioid receptors with the specific μ-opioid receptor antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-PenThr-NH2 (1 ng CTAP) within the NAc shell had no effect in either sex. Thus, κ-opioid receptors within the NAc shell mediate aversive social motivation that is critical for pair bond maintenance. PMID:22593047

  8. 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

  9. 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.

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

    PubMed Central

    Banker, D E; Eisenman, R N

    1993-01-01

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

  11. Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor

    PubMed Central

    Callaghan, Brid; Kosari, Samin; Pustovit, Ruslan V; Sartor, Daniela M; Ferens, Dorota; Ban, Kung; Baell, Jonathan; Nguyen, Trung V; Rivera, Leni R; Brock, James A; Furness, John B

    2014-01-01

    BACKGROUND AND PURPOSE Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. PMID:24670149

  12. Dynamics of receptor-mediated nanoparticle internalization into endothelial cells.

    PubMed

    Gonzalez-Rodriguez, David; Barakat, Abdul I

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process.

  13. Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

    PubMed Central

    Van Amersfoort, Edwin S.; Van Berkel, Theo J. C.; Kuiper, Johan

    2003-01-01

    Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins. PMID:12857774

  14. An histidine covalent receptor and butenolide complex mediates strigolactone perception.

    PubMed

    de Saint Germain, Alexandre; Clavé, Guillaume; Badet-Denisot, Marie-Ange; Pillot, Jean-Paul; Cornu, David; Le Caer, Jean-Pierre; Burger, Marco; Pelissier, Frank; Retailleau, Pascal; Turnbull, Colin; Bonhomme, Sandrine; Chory, Joanne; Rameau, Catherine; Boyer, François-Didier

    2016-10-01

    Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

  15. An histidine covalent receptor and butenolide complex mediates strigolactone perception.

    PubMed

    de Saint Germain, Alexandre; Clavé, Guillaume; Badet-Denisot, Marie-Ange; Pillot, Jean-Paul; Cornu, David; Le Caer, Jean-Pierre; Burger, Marco; Pelissier, Frank; Retailleau, Pascal; Turnbull, Colin; Bonhomme, Sandrine; Chory, Joanne; Rameau, Catherine; Boyer, François-Didier

    2016-10-01

    Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand. PMID:27479744

  16. Functional profile of the binary brain corticosteroid receptor system: mediating, multitasking, coordinating, integrating.

    PubMed

    de Kloet, E R

    2013-11-01

    This contribution is focused on the action of the naturally occurring corticosteroids, cortisol and corticosterone, which are secreted from the adrenals in hourly pulses and after stress with the goal to maintain resilience and health. To achieve this goal the action of the corticosteroids displays an impressive diversity, because it is cell-specific and context-dependent in coordinating the individual's response to changing environments. These diverse actions of corticosterone are mediated by mineralocorticoid- and glucocorticoid-receptors that operate as a binary system in concert with neurotransmitter and neuropeptide signals to activate and inhibit stress reactions, respectively. Classically MR and GR are gene transcription factors, but recently these receptors appear to mediate also rapid non-genomic actions on excitatory neurotransmission suggesting that they integrate functions over time. Hence the balance of receptor-mediated actions is crucial for homeostasis. This balanced function of mineralo- and glucocorticoid-receptors can be altered epigenetically by a history of traumatic (early) life events and the experience of repeated stressors as well as by predisposing genetic variants in signaling pathways of these receptors. One of these variants, mineralocorticoid receptor haplotype 2, is associated with dispositional optimism in appraisal of environmental challenges. Imbalance in receptor-mediated corticosterone actions was found to leave a genomic signature highlighting the role of master switches such as cAMP response element-binding protein and mammalian target of rapamycin to compromise health, and to promote vulnerability to disease. Diabetic encephalopathy is a pathology of imbalanced corticosterone action, which can be corrected in its pre-stage by a brief treatment with the antiglucocorticoid mifepristone.

  17. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  18. 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.

  19. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

    PubMed Central

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  20. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

  1. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  2. Scavenger receptor-mediated endocytosis by sinusoidal cells in rat bone marrow

    SciTech Connect

    Geoffroy, J.S.

    1987-01-01

    Endocytosis of serum albumin by sinusoidal endothelial cells in rat bone marrow was investigated initially at the ultrastructural level with subsequent biochemical investigation of the specificity mediating this event. Bovine serum albumin adsorbed to 20nm colloidal gold particles (AuBSA) was chosen as the electron microscopic probe. Morphological data strongly suggested that a receptor was involved in uptake of AuBSA. Confirmation of receptor involvement in the uptake of AuBSA by marrow sinusoidal endothelial cells was achieved utilizing an in situ isolated hind limb perfusion protocol in conjunction with unlabeled, radiolabeled, and radio-/colloidal gold labeled probes. The major findings of competition and saturation experiments were: (1) endocytosis of AuBSA was mediated by a receptor for modified/treated serum albumin; (2) endocytosis of formaldehyde-treated serum albumin was mediated by a binding site which may be the same or closely related to the site responsible for the uptake of AuBSA; and (3) endocytosis of native untreated albumin was not mediated by receptor and probably represents fluid-phase pinocitosis.

  3. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  4. 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

  5. Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway.

    PubMed

    Zhu, Ping; Baek, Sung Hee; Bourk, Eliot M; Ohgi, Kenneth A; Garcia-Bassets, Ivan; Sanjo, Hideki; Akira, Shizuo; Kotol, Paul F; Glass, Christopher K; Rosenfeld, Michael G; Rose, David W

    2006-02-10

    Defining the precise molecular strategies that coordinate patterns of transcriptional responses to specific signals is central for understanding normal development and homeostasis as well as the pathogenesis of hormone-dependent cancers. Here we report specific prostate cancer cell/macrophage interactions that mediate a switch in function of selective androgen receptor antagonists/modulators (SARMs) from repression to activation in vivo. This is based on an evolutionarily conserved receptor N-terminal L/HX7LL motif, selectively present in sex steroid receptors, that causes recruitment of TAB2 as a component of an N-CoR corepressor complex. TAB2 acts as a sensor for inflammatory signals by serving as a molecular beacon for recruitment of MEKK1, which in turn mediates dismissal of the N-CoR/HDAC complex and permits derepression of androgen and estrogen receptor target genes. Surprisingly, this conserved sensor strategy may have arisen to mediate reversal of sex steroid-dependent repression of a limited cohort of target genes in response to inflammatory signals, linking inflammatory and nuclear receptor ligand responses to essential reproductive functions.

  6. Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

    PubMed

    Celik, Melih Ö; Labuz, Dominika; Henning, Karen; Busch-Dienstfertig, Melanie; Gaveriaux-Ruff, Claire; Kieffer, Brigitte L; Zimmer, Andreas; Machelska, Halina

    2016-10-01

    Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the G

  7. Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

    PubMed

    Celik, Melih Ö; Labuz, Dominika; Henning, Karen; Busch-Dienstfertig, Melanie; Gaveriaux-Ruff, Claire; Kieffer, Brigitte L; Zimmer, Andreas; Machelska, Halina

    2016-10-01

    Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the G

  8. Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture

    PubMed Central

    McDonald, John L.; Cripps, Allan W.; Smith, Peter K.

    2015-01-01

    Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1), a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture. PMID:26339274

  9. Fc alpha receptors mediate release of tumour necrosis factor-alpha and interleukin-6 by human monocytes following receptor aggregation.

    PubMed Central

    Patry, C; Herbelin, A; Lehuen, A; Bach, J F; Monteiro, R C

    1995-01-01

    The functional capacity of the human monocyte receptor for the Fc portion of IgA (Fc alpha R) in mediating signal transduction was evaluated by cytokine release. F(ab')2 fragments of anti-Fc alpha R monoclonal antibodies (mAb) were used as specific probes to induce release of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Multivalent cross-linking by a secondary anti-mouse antibody [F(ab')2 fragments] induced a significant release of TNF-alpha and IL-6 by human blood mononuclear cells, indicating requirements for Fc alpha R aggregation on the cell surface to transmit signals. Both cytokines were released exclusively by adherent cells, identifying monocytes as the responding cells within the mononuclear cell population. This cytokine release could not be due to contaminating endotoxins, because it was not abolished by polymyxin B, a lipopolysaccharide (LPS) inhibitor. Moreover, purified recombinant soluble Fc alpha R inhibited the anti-Fc alpha R mAb-mediated cytokine release from blood monocytes, demonstrating that TNF-alpha and IL-6 were released in a receptor-specific manner. Our data suggest that Fc alpha R, through its capacity to mediate secretion of IL-6, may play an important role in B-cell proliferation and immunoglobulin production. On the other hand, release of TNF-alpha following stimulation of Fc alpha R molecules directly implicates these receptors in amplification and regulation of the inflammatory process occurring during IgA-mediated host defence. PMID:7590867

  10. Cannabinoid receptor 1 is a major mediator of renal fibrosis.

    PubMed

    Lecru, Lola; Desterke, Christophe; Grassin-Delyle, Stanislas; Chatziantoniou, Christos; Vandermeersch, Sophie; Devocelle, Aurore; Vernochet, Amelia; Ivanovski, Ninoslav; Ledent, Catherine; Ferlicot, Sophie; Dalia, Meriem; Saïd, Myriam; Beaudreuil, Séverine; Charpentier, Bernard; Vazquez, Aimé; Giron-Michel, Julien; Azzarone, Bruno; Durrbach, Antoine; François, Hélène

    2015-07-01

    Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

  11. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors

    PubMed Central

    Mrozkova, Petra; Spicarova, Diana; Palecek, Jiri

    2016-01-01

    Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception. PMID:27755539

  12. NPY/Y1 receptor-mediated vasoconstrictory and proliferative effects in pulmonary hypertension

    PubMed Central

    Crnkovic, S; Egemnazarov, B; Jain, P; Seay, U; Gattinger, N; Marsh, L M; Bálint, Z; Kovacs, G; Ghanim, B; Klepetko, W; Schermuly, R T; Weissmann, N; Olschewski, A; Kwapiszewska, G

    2014-01-01

    BACKGROUND AND PURPOSE Pulmonary arteries (PAs) are innervated, but little is known about the role of neuronal axis in pulmonary hypertension (PH). Here, we have examined the role of the neuropeptide Y (NPY) and its Y1 receptor in PH pathogenesis. EXPERIMENTAL APPROACH NPY was localized by immunofluorescence. Expression of NPY and Y1 receptor were determined by quantitative PCR. Cellular response to NPY stimulation was assessed by Western blotting, thymidine incorporation and calcium imaging. Wire myography and isolated perfused mouse lung were applied to study pulmonary vasoactive effects of NPY. Selective receptor antagonists were used to assess the contribution of receptor subtypes in mediating NPY effects. KEY RESULTS Samples from PH patients showed increased NPYergic innervation within the PA wall and higher Y1 receptor expression, compared with donors. However, NPY levels were unchanged in both PA and serum. In the chronic hypoxic mouse model, Y1 receptor were up-regulated, while expression of both NPY and Y1 receptor was increased in the lungs of monocrotaline and SU5416-hypoxia rats. On a functional level, NPY acutely increased intracellular calcium levels and enhanced vasoconstriction of lung vessels preconstricted with adrenaline. Furthermore, NPY stimulated proliferation of human pulmonary arterial smooth muscle cells and activated p38 and PKD pathways. Correspondingly, higher phosphorylation of PKD was observed in remodelled vessels from PH patients. The selective Y1 receptor antagonist, BIBO 3304, concentration-dependently inhibited vasoconstrictive and proliferative effects of NPY. CONCLUSIONS AND IMPLICATIONS NPY and Y1 receptor are possible mediators of both vasoconstriction and pulmonary vascular remodelling in PH. PMID:24779394

  13. 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

  14. 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.

  15. Hippocampus NMDA receptors selectively mediate latent extinction of place learning.

    PubMed

    Goodman, Jarid; Gabriele, Amanda; Packard, Mark G

    2016-09-01

    Extinction of maze learning may be achieved with or without the animal performing the previously acquired response. In typical "response extinction," animals are given the opportunity to make the previously acquired approach response toward the goal location of the maze without reinforcement. In "latent extinction," animals are not given the opportunity to make the previously acquired response and instead are confined to the previous goal location without reinforcement. Previous evidence indicates that the effectiveness of these protocols may depend on the type of memory being extinguished. Thus, one aim of the present study was to further examine the effectiveness of response and latent extinction protocols across dorsolateral striatum (DLS)-dependent response learning and hippocampus-dependent place learning tasks. In addition, previous neural inactivation experiments indicate a selective role for the hippocampus in latent extinction, but have not investigated the precise neurotransmitter mechanisms involved. Thus, the present study also examined whether latent extinction of place learning might depend on NMDA receptor activity in the hippocampus. In experiment 1, adult male Long-Evans rats were trained in a response learning task in a water plus-maze, in which animals were reinforced to make a consistent body-turn response to reach an invisible escape platform. Results indicated that response extinction, but not latent extinction, was effective at extinguishing memory in the response learning task. In experiment 2, rats were trained in a place learning task, in which animals were reinforced to approach a consistent spatial location containing the hidden escape platform. In experiment 2, animals also received intra-hippocampal infusions of the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid (AP5; 5.0 or 7.5 ug/0.5 µg) or saline vehicle immediately before response or latent extinction training. Results indicated that both extinction protocols were

  16. Hippocampus NMDA receptors selectively mediate latent extinction of place learning.

    PubMed

    Goodman, Jarid; Gabriele, Amanda; Packard, Mark G

    2016-09-01

    Extinction of maze learning may be achieved with or without the animal performing the previously acquired response. In typical "response extinction," animals are given the opportunity to make the previously acquired approach response toward the goal location of the maze without reinforcement. In "latent extinction," animals are not given the opportunity to make the previously acquired response and instead are confined to the previous goal location without reinforcement. Previous evidence indicates that the effectiveness of these protocols may depend on the type of memory being extinguished. Thus, one aim of the present study was to further examine the effectiveness of response and latent extinction protocols across dorsolateral striatum (DLS)-dependent response learning and hippocampus-dependent place learning tasks. In addition, previous neural inactivation experiments indicate a selective role for the hippocampus in latent extinction, but have not investigated the precise neurotransmitter mechanisms involved. Thus, the present study also examined whether latent extinction of place learning might depend on NMDA receptor activity in the hippocampus. In experiment 1, adult male Long-Evans rats were trained in a response learning task in a water plus-maze, in which animals were reinforced to make a consistent body-turn response to reach an invisible escape platform. Results indicated that response extinction, but not latent extinction, was effective at extinguishing memory in the response learning task. In experiment 2, rats were trained in a place learning task, in which animals were reinforced to approach a consistent spatial location containing the hidden escape platform. In experiment 2, animals also received intra-hippocampal infusions of the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid (AP5; 5.0 or 7.5 ug/0.5 µg) or saline vehicle immediately before response or latent extinction training. Results indicated that both extinction protocols were

  17. PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons.

    PubMed

    Yang, D-J; Wang, X-L; Ismail, A; Ashman, C J; Valori, C F; Wang, G; Gao, S; Higginbottom, A; Ince, P G; Azzouz, M; Xu, J; Shaw, P J; Ning, K

    2014-02-27

    Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.

  18. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  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. Duloxetine Inhibits Microglial P2X4 Receptor Function and Alleviates Neuropathic Pain after Peripheral Nerve Injury

    PubMed Central

    Yamashita, Tomohiro; Yamamoto, Shota; Zhang, Jiaming; Kometani, Miho; Tomiyama, Daisuke; Kohno, Keita; Tozaki-Saitoh, Hidetoshi; Inoue, Kazuhide; Tsuda, Makoto

    2016-01-01

    P2X4 receptors (P2X4R) are a family of ATP-gated non-selective cation channels. We previously demonstrated that activation of P2X4R in spinal microglia is crucial for neuropathic pain, a highly debilitating chronic pain condition, suggesting that P2X4R is a potential therapeutic target for treating neuropathic pain. Thus, the identification of a compound that has a potent inhibitory effect on P2X4R is an important clinical challenge. In the present study, we screened a chemical library of clinically approved drugs and show for the first time that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rodent and human P2X4R. In primary cultured microglial cells, duloxetine also inhibited P2X4R-, but not P2X7R-, mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain produced a reversal of nerve injury-induced mechanical allodynia, a cardinal symptom of neuropathic pain. In rats that were pretreated with a serotonin-depleting agent and a noradrenaline neurotoxin, the antiallodynic effect of duloxetine was reduced, but still remained. Based on these results, we suggest that, in addition to duloxetine’s primary inhibitory action on serotonin and noradrenaline transporters, an inhibitory effect on P2X4R may be involved at least in part in an antiallodynic effect of intrathecal duloxetine in a model of neuropathic pain. PMID:27768754

  1. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  2. GSG1L suppresses AMPA receptor-mediated synaptic transmission and uniquely modulates AMPA receptor kinetics in hippocampal neurons.

    PubMed

    Gu, Xinglong; Mao, Xia; Lussier, Marc P; Hutchison, Mary Anne; Zhou, Liang; Hamra, F Kent; Roche, Katherine W; Lu, Wei

    2016-01-01

    Regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is a key mechanism for synaptic plasticity. In the brain, AMPARs assemble with a number of auxiliary subunits, including TARPs, CNIHs and CKAMP44, which are important for AMPAR forward trafficking to synapses. Here we report that the membrane protein GSG1L negatively regulates AMPAR-mediated synaptic transmission. Overexpression of GSG1L strongly suppresses, and GSG1L knockout (KO) enhances, AMPAR-mediated synaptic transmission. GSG1L-dependent regulation of AMPAR synaptic transmission relies on the first extracellular loop domain and its carboxyl-terminus. GSG1L also speeds up AMPAR deactivation and desensitization in hippocampal CA1 neurons, in contrast to the effects of TARPs and CNIHs. Furthermore, GSG1L association with AMPARs inhibits CNIH2-induced slowing of the receptors in heterologous cells. Finally, GSG1L KO rats have deficits in LTP and show behavioural abnormalities in object recognition tests. These data demonstrate that GSG1L represents a new class of auxiliary subunit with distinct functional properties for AMPARs. PMID:26932439

  3. GSG1L suppresses AMPA receptor-mediated synaptic transmission and uniquely modulates AMPA receptor kinetics in hippocampal neurons

    PubMed Central

    Gu, Xinglong; Mao, Xia; Lussier, Marc P.; Hutchison, Mary Anne; Zhou, Liang; Hamra, F. Kent; Roche, Katherine W.; Lu, Wei

    2016-01-01

    Regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is a key mechanism for synaptic plasticity. In the brain, AMPARs assemble with a number of auxiliary subunits, including TARPs, CNIHs and CKAMP44, which are important for AMPAR forward trafficking to synapses. Here we report that the membrane protein GSG1L negatively regulates AMPAR-mediated synaptic transmission. Overexpression of GSG1L strongly suppresses, and GSG1L knockout (KO) enhances, AMPAR-mediated synaptic transmission. GSG1L-dependent regulation of AMPAR synaptic transmission relies on the first extracellular loop domain and its carboxyl-terminus. GSG1L also speeds up AMPAR deactivation and desensitization in hippocampal CA1 neurons, in contrast to the effects of TARPs and CNIHs. Furthermore, GSG1L association with AMPARs inhibits CNIH2-induced slowing of the receptors in heterologous cells. Finally, GSG1L KO rats have deficits in LTP and show behavioural abnormalities in object recognition tests. These data demonstrate that GSG1L represents a new class of auxiliary subunit with distinct functional properties for AMPARs. PMID:26932439

  4. 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.

  5. Contractions of the mouse prostate elicited by acetylcholine are mediated by M(3) muscarinic receptors.

    PubMed

    White, Carl W; Short, Jennifer L; Haynes, John M; Matsui, Minoru; Ventura, Sabatino

    2011-12-01

    Increased smooth muscle tone in the human prostate contributes to the symptoms associated with benign prostatic hyperplasia. In the mouse prostate gland, cholinergic innervation is responsible for a component of the nerve-mediated contractile response. This study investigates the muscarinic receptor subtype responsible for the cholinergic contractile response in the mouse prostate gland. To characterize the muscarinic receptor subtype, mouse prostates taken from wild-type or M(3) muscarinic receptor knockout mice were mounted in organ baths. The isometric force that tissues developed in response to electrical-field stimulation or exogenously applied cholinergic agonists in the presence or absence of a range of muscarinic receptor antagonists was evaluated. Carbachol elicited reproducible and concentration-dependent contractions of the isolated mouse prostate, which were antagonized by the presence of muscarinic receptor antagonists. Calculation of antagonist affinities (pA(2) values) indicated a rank order of antagonist potencies in the mouse prostate of: darifenacin (9.08) = atropine (9.07) = 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (9.02) > cyclohexyl-hydroxy-phenyl-(3-piperidin-1-ylpropyl)silane (7.85) > cyclohexyl-(4-fluorophenyl)-hydroxy-(3-piperidin-1-ylpropyl)silane (7.39) > himbacine (7.19) > pirenzipine (6.88) > methoctramine (6.20). Furthermore, genetic deletion of the M(3) muscarinic receptor inhibited prostatic contractions to electrical-field stimulation or exogenous administration of acetylcholine. In this study we identified that the cholinergic component of contraction in the mouse prostate is mediated by the M(3) muscarinic receptor subtype. Pharmacological antagonism of the M(3) muscarinic receptor may be a beneficial additional target for the treatment of benign prostatic hyperplasia in the human prostate gland.

  6. Sphingosine 1-phosphate receptors are essential mediators of eyelid closure during embryonic development.

    PubMed

    Herr, Deron R; Lee, Chang-Wook; Wang, Wei; Ware, Adam; Rivera, Richard; Chun, Jerold

    2013-10-11

    The fetal development of the mammalian eyelid involves the expansion of the epithelium over the developing cornea, fusion into a continuous sheet covering the eye, and a splitting event several weeks later that results in the formation of the upper and lower eyelids. Recent studies have revealed a significant number of molecular signaling components that are essential mediators of eyelid development. Receptor-mediated sphingosine 1-phosphate (S1P) signaling is known to influence diverse biological processes, but its involvement in eyelid development has not been reported. Here, we show that two S1P receptors, S1P2 and S1P3, are collectively essential mediators of eyelid closure during murine development. Homozygous deletion of the gene encoding either receptor has no apparent effect on eyelid development, but double-null embryos are born with an "eyes open at birth" defect due to a delay in epithelial sheet extension. Both receptors are expressed in the advancing epithelial sheet during the critical period of extension. Fibroblasts derived from double-null embryos have a deficient response to epidermal growth factor, suggesting that S1P2 and S1P3 modulate this essential signaling pathway during eyelid closure.

  7. Molecular mechanisms involving sigma receptor-mediated induction of MCP-1: implication for increased monocyte transmigration.

    PubMed

    Yao, Honghong; Yang, Yanjing; Kim, Kee Jun; Bethel-Brown, Crystal; Gong, Nan; Funa, Keiko; Gendelman, Howard E; Su, Tsung-Ping; Wang, John Q; Buch, Shilpa

    2010-06-10

    Cocaine abuse hastens the neurodegeneration often associated with advanced HIV-1 infection. The mechanisms, in part, revolve around the neuroinflammatory processes mediated by the chemokine monocyte chemotactic protein-1 (MCP-1/CCL2). Understanding factors that modulate MCP-1 and, in turn, facilitate monocyte extravasation in the brain is thus of paramount importance. We now demonstrate that cocaine induces MCP-1 in rodent microglia through translocation of the sigma receptor to the lipid raft microdomains of the plasma membrane. Sequential activation of Src, mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3' kinase (PI3K)/Akt and nuclear factor kappaB (NF-kappaB) pathways resulted in increased MCP-1 expression. Furthermore, conditioned media from cocaine-exposed microglia increased monocyte transmigration, and thus was blocked by antagonists for CCR2 or sigma receptor. These findings were corroborated by demonstrating increased monocyte transmigration in mice exposed to cocaine, which was attenuated by pretreatment of mice with the sigma receptor antagonist. Interestingly, cocaine-mediated transmigratory effects were not observed in CCR2 knockout mice. We conclude that cocaine-mediated induction of MCP-1 accelerates monocyte extravasation across the endothelium. Understanding the regulation of MCP-1 expression and functional changes by cocaine/sigma receptor system may provide insights into the development of potential therapeutic targets for HIV-1-associated neurocognitive disorders. PMID:20354174

  8. 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.

  9. beta-Arrestin mediates beta1-adrenergic receptor-epidermal growth factor receptor interaction and downstream signaling.

    PubMed

    Tilley, Douglas G; Kim, Il-Man; Patel, Priyesh A; Violin, Jonathan D; Rockman, Howard A

    2009-07-24

    beta1-Adrenergic receptor (beta1AR) stimulation confers cardioprotection via beta-arrestin-de pend ent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the beta1AR and EGFR form a complex that differentially directs intracellular signaling pathways. beta1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas beta1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. beta1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of beta1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of beta1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the beta1AR and recruitment of beta-arrestin. These data reveal a new signaling paradigm in which beta-arrestin is required for the maintenance of a beta1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

  10. 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

  11. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

    PubMed Central

    Woodier, Jason; Rainbow, Richard D.; Stewart, Alan J.; Pitt, Samantha J.

    2015-01-01

    Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  12. Ghrelin-induced hypophagia is mediated by the β2 adrenergic receptor in chicken.

    PubMed

    Zendehdel, Morteza; Hassanpour, Shahin

    2014-09-01

    The purpose of this study was to examine the effects of intracerebroventricular injection of metoprolol (a β1 adrenergic receptor antagonist), ICI 118,551 (a β2 adrenergic receptor antagonist), and SR 59230R (a β3 adrenergic receptor antagonist) on ghrelin-induced food and water intake by 3-h food-deprived (FD3) cockerels. The chickens were randomly allocated to 4 treatment groups with 8 replicates in each group. A cannula was surgically implanted into the lateral ventricle of the brain. In experiment 1, chickens received the β1 adrenergic receptor antagonist (24 nmol) before injection of the ghrelin (0.6 nmol). In experiment 2, chickens received the β2 adrenergic receptor antagonist (5 nmol) before injection of the ghrelin (0.6 nmol). In experiment 3, birds were injected with ghrelin (0.6 nmol) after the β3 adrenergic receptor antagonist (20 nmol). Cumulative food and water intake were recorded 3-h post injection and analyzed by two-way analysis of variance. According to the results, ghrelin injection reduced food and water intake by broiler cockerels (p≤0.05). The effect of ghrelin on food intake was significantly attenuated by pretreatment with the β2 receptor antagonist (p≤0.05). Furthermore, the β2 receptor antagonist had no effect on water intake induced by ghrelin. Also, pretreatment with the β1 and β3 receptors antagonists had no effect on ghrelin-induced food and water intake. These results suggest that the effect of ghrelin on cumulative food intake by cockerels is mediated via β2 adrenergic receptors.

  13. NOP receptor mediates anti-analgesia induced by agonist-antagonist opioids.

    PubMed

    Gear, R W; Bogen, O; Ferrari, L F; Green, P G; Levine, J D

    2014-01-17

    Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ∼90min after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J-113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

  14. IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

    PubMed

    Braun, Matthias; Ress, Marie L; Yoo, Young-Eun; Scholz, Claus J; Eyrich, Matthias; Schlegel, Paul G; Wölfl, Matthias

    2016-07-01

    Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1. PMID:27622043

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

    PubMed

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

    2014-12-26

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

  16. 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

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

    PubMed

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

    2014-12-26

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

  18. 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.

  19. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  20. Sex mediates dopamine and adrenergic receptor expression in adult rats exposed prenatally to cocaine

    PubMed Central

    Ferris, Mark J.; Mactutus, Charles F.; Silvers, Janelle M.; Hasselrot, Ulla; Strupp, Barbara J.; Booze, Rosemarie M.

    2010-01-01

    The extent of catecholaminergic receptor and respective behavioral alterations associated with prenatal cocaine exposure varies according to exogenous factors such as the amount, frequency, and route of maternal exposure, as well as endogenous factors such as specific brain regions under consideration and sex of the species. The goal of the current study was to use autoradiography to delineate possible moderators of dopaminergic and adrenergic receptor expression in adult rat offspring exposed to cocaine in utero. The current study demonstrated sex-dependent D1 receptor, α2, and noradrenergic transporter binding alterations in prelimbic, hippocampus, and anterior cingulate regions of adult rat brains exposed to cocaine during gestational days 8–21. Of further interest was the lack of alterations in the nucleus accumbens for nearly all receptors/transporters investigated, as well as the lack of alterations in D3 receptor binding in nearly all of the regions investigated (nucleus accumbens, prelimbic region, hippocampus, and cingulate gyrus). Thus, the current investigation demonstrated persistent receptor and transporter alterations that extend well into adulthood as a result of cocaine exposure in utero. Furthermore, the demonstration that sex played a mediating role in prenatal cocaine-induced, aberrant receptor/transporter expression is of primary importance for future studies that seek to control for sex in either design or analysis. PMID:17933484

  1. Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor.

    PubMed Central

    Abu-Amer, Y; Ross, F P; Edwards, J; Teitelbaum, S L

    1997-01-01

    Chronic bone infection, as attends periodontitis, is often complicated by severe osteolysis. While LPS is believed to be central to the pathogenesis of the osteolytic lesion, the mechanisms by which this bacteria-derived molecule promotes bone resorption are unknown. We find that LPS induces bone marrow macrophages (BMMs) to express c-src, a protooncogene product that we demonstrate is a specific marker of commitment to the osteoclast phenotype. We next turned to possible soluble mediators of LPS-induced c-src. Of a number of osteoclastogenic cytokines tested, only TNF-alpha mirrors the c-src-enhancing effect of LPS. Suggesting that LPS augmentation of c-src is TNF-mediated, endotoxin sequentially induces BMM expression of TNF, followed by c-src. TNF and c-src expression, by cultured BMMs derived from LPS-injected mice, reflects duration of exposure to circulating endotoxin, intimating that endotoxin's effect in vivo is also mediated by TNF. Consistent with these findings, thalidomide (which antagonizes TNF action) attenuates c-src induction by LPS. An anti-TNF antibody blocks LPS enhancement of c-src mRNA, validating the cytokine's modulating role in vitro. Using BMMs of TNF receptor-deleted mice, we demonstrate that TNF induction of c-src is transmitted through the cytokine's p55, but not p75, receptor. Most importantly, LPS administered to wild-type mice prompts osteoclast precursor differentiation, manifest by profound osteoclastogenesis in marrow cultured ex vivo, and by a profusion of marrow-residing cells expressing the osteoclast marker tartrate resistant acid phosphatase, in vivo. In contrast, LPS does not substantially enhance osteoclast proliferation in mice lacking the p55TNF receptor, confirming that LPS-induced osteoclastogenesis is mediated by TNF in vivo via this receptor. Thus, therapy targeting TNF and/or its p55 receptor presents itself as a means of preventing the osteolysis of chronic bacterial infection. PMID:9294124

  2. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

    PubMed Central

    Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

    2011-01-01

    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

  3. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.

    PubMed

    Murphy, Jane E; Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Feld, Micha; Brand, Eva; Cedron, Wendy J; Bunnett, Nigel W; Steinhoff, Martin

    2011-10-01

    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

  4. Brain stem adenosine receptors modulate centrally mediated hypotensive responses in conscious rats: A review.

    PubMed

    Nassar, Noha N; Abdel-Rahman, Abdel A

    2015-05-01

    Adenosine is implicated in the modulation of cardiovascular responses either at the peripheral or at central level in experimental animals. However, there are no dedicated reviews on the involvement of adenosine in mediating the hypotensive response of centrally administered clonidine in general and specifically in aortically barodenervated rats (ABD). The conscious ABD rat model exhibits surgically induced baroreflex dysfunction and exaggerated hypotensive response, compared with conscious sham-operated (SO) rats. The current review focuses on, the role of adenosine receptors in blood pressure (BP) regulation and their possible crosstalk with other receptors e.g. imidazoline (I1) and alpha (α2A) adrenergic receptor (AR). The former receptor is a molecular target for clonidine, whose hypotensive effect is enhanced approx. 3-fold in conscious ABD rats. We also discussed how the balance between the brain stem adenosine A1 and A2A receptors is regulated by baroreceptors and how such balance influences the centrally mediated hypotensive responses. The use of the ABD rat model yielded insight into the downstream signaling cascades following clonidine-evoked hypotension in a surgical model of baroreflex dysfunction. PMID:26257930

  5. Dual role of dopamine D(2)-like receptors in the mediation of conditioned and unconditioned fear.

    PubMed

    Brandão, Marcus Lira; de Oliveira, Amanda Ribeiro; Muthuraju, Sangu; Colombo, Ana Caroline; Saito, Viviane Mitsuko; Talbot, Teddy

    2015-11-14

    A reduction of dopamine release or D2 receptor blockade in the terminal fields of the mesolimbic system, particularly the amygdala, clearly reduces conditioned fear. Similar D2 receptor antagonism in the neural substrates of fear in the midbrain tectum attenuates the processing of unconditioned aversive information. However, the implications of the interplay between opposing actions of dopamine in the rostral and caudal segments of the dopaminergic system are still unclear. Previous studies from this laboratory have reported the effects of dopaminergic drugs on behavior in rats in the elevated plus maze, auditory-evoked potentials (AEPs) recorded from the midbrain tectum, fear-potentiated startle, and conditioned freezing. These findings led to an interesting framework on the functional roles of dopamine in both anxiety and fear states. Dopamine D2 receptor inhibition in the terminal fields of the mesolimbic dopamine system generally causes anxiolytic-like effects, whereas the activity of midbrain substrates of unconditioned fear are enhanced by D2 receptor antagonists, suggesting that D2 receptor-mediated mechanisms play opposing roles in fear/anxiety processes, depending on the brain region under study. Dopamine appears to mediate conditioned fear by acting at rostral levels of the brain and regulate unconditioned fear at the midbrain level, likely by reducing the sensorimotor gating of aversive events.

  6. 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.

  7. 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.

  8. 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.

  9. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  10. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies.

    PubMed

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-01-01

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects. PMID:27412401

  11. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies

    PubMed Central

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-01-01

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects. PMID:27412401

  12. 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.

  13. 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

  14. 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.

  15. Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions

    SciTech Connect

    Steranka, L.R.; Manning, D.C.; DeHaas, C.J.; Ferkany, J.W.; Borosky, S.A.; Connor, J.R.; Vavrek, R.J.; Stewart, J.M.; Snyder, S.H.

    1988-05-01

    Autoradiographic studies localize (/sup 3/H)bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. (/sup 3/H)Bradykinin labeling is also observed over myocardinal/coronary visceral afferent fibers. The localization of (/sup 3/H)bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradkykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.

  16. 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.

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

    PubMed

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

    1999-08-11

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

  18. 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.

  19. 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.

  20. New Insights into VacA Intoxication Mediated through Its Cell Surface Receptors

    PubMed Central

    Yahiro, Kinnosuke; Hirayama, Toshiya; Moss, Joel; Noda, Masatoshi

    2016-01-01

    Helicobacter pylori (H. pylori), a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPβ, and LRP1) that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin) have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage. PMID:27187473

  1. Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation.

    PubMed

    Soriano, Silvia F; Hernanz-Falcón, Patricia; Rodríguez-Frade, José Miguel; De Ana, Ana Martín; Garzón, Ruth; Carvalho-Pinto, Carla; Vila-Coro, Antonio J; Zaballos, Angel; Balomenos, Dimitrios; Martínez-A, Carlos; Mellado, Mario

    2002-08-01

    Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca(2+) flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Galpha(i) pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.

  2. Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila

    PubMed Central

    Knecht, Zachary A; Silbering, Ana F; Ni, Lina; Klein, Mason; Budelli, Gonzalo; Bell, Rati; Abuin, Liliane; Ferrer, Anggie J; Samuel, Aravinthan DT; Benton, Richard; Garrity, Paul A

    2016-01-01

    Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most evolutionarily deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is required for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects. DOI: http://dx.doi.org/10.7554/eLife.17879.001 PMID:27656904

  3. Modeling of cell adhesion and deformation mediated by receptor-ligand interactions.

    PubMed

    Golestaneh, Amirreza F; Nadler, Ben

    2016-04-01

    The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor-ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor-ligand interaction via Fick's Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell.

  4. Modeling of cell adhesion and deformation mediated by receptor-ligand interactions.

    PubMed

    Golestaneh, Amirreza F; Nadler, Ben

    2016-04-01

    The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor-ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor-ligand interaction via Fick's Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell. PMID:26093646

  5. Characterization of the 5-hydroxytryptamine receptor which mediates contraction of the human isolated basilar artery.

    PubMed

    Parsons, A A; Whalley, E T

    1989-01-01

    This paper reports part of a study which investigated the identity of the receptor involved in 5-hydroxytryptamine (5-HT) mediated contraction of the human basilar artery in vitro. 5-HT and a variety of 5-HT receptor agonists contracted human isolated basilar artery with a rank order of agonist potency: 5-carboxamidotryptamine greater than 5-HT greater than GR43175 much much greater than 2-methyl-5-HT. The maximum response produced by these agonists differed. The contractile responses to both 5-HT and GR43175 were resistant to antagonism by the 5-HT2 antagonist ketanserin and the 5-HT3 antagonist GR38032, indicating that neither 5-HT nor GR43175 activate 5-HT2 and 5-HT3 receptors in this tissue. In striking contrast, methiothepin (an antagonist at 5-HT1-like receptors) proved a potent antagonist of the contractile actions of both 5-HT and GR43175. Methiothepin did not antagonize the contractile response to the thromboxane-A2 mimetic U-46619. It is concluded that 5-HT and GR43175 contract the human isolated basilar artery by activating 5-HT1-like receptors. It is suggested that the antimigraine action of GR43175 may reflect its ability to constrict certain cranial arteries via 5-HT1-like receptor activation. PMID:2544283

  6. Augmentation of Antigen Receptor–mediated Responses by Histamine H1 Receptor Signaling

    PubMed Central

    Banu, Yasmin; Watanabe, Takeshi

    1999-01-01

    Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein–coupled receptors. Here, we report that histamine may affect antigen receptor–mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3ε– or anti-IgM–stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit–positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3ε cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor–mediated immune responses, suggesting cross-talk between G protein–coupled receptors and antigen receptor–mediated signaling. PMID:9989982

  7. Toll-like receptor 4 deficiency impairs microglial phagocytosis of degenerating axons.

    PubMed

    Rajbhandari, Labchan; Tegenge, Million Adane; Shrestha, Shiva; Ganesh Kumar, Nishant; Malik, Adeel; Mithal, Aditya; Hosmane, Suneil; Venkatesan, Arun

    2014-12-01

    Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration.

  8. 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.

  9. 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

  10. 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.

  11. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

    PubMed

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

    2012-04-13

    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex. PMID:22375000

  12. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

    PubMed

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

    2012-04-13

    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

  13. B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.

    PubMed Central

    Westermark, B; Siegbahn, A; Heldin, C H; Claesson-Welsh, L

    1990-01-01

    Porcine aorta endothelial cells are devoid of receptors for platelet-derived growth factor (PDGF). We have transfected such cells with cDNA for the PDGF B-type receptor, both the wild-type receptor and a mutant form of the receptor (K634A), in which the putative nucleotide-binding lysine of the protein-tyrosine domain has been changed to alanine. Immunoprecipitation studies of metabolically labeled cells showed that both types of receptors were synthesized and processed to the mature form of Mr 190,000. In cells expressing the wild-type receptor, PDGF-BB, the natural ligand for the B-type receptor, induced membrane ruffling and reorganization of actin. Such a response has previously been seen in cells expressing the natural PDGF B-type receptor in response to PDGF-BB. No such effect was induced in nontransfected cells or in cells expressing the K634A mutant receptor. PDGF was also shown to be chemotactic for cells expressing the wild-type receptor, whereas no chemotactic response was elicited in control cells or in cells expressing the K634A mutant receptor. Our study thus provides formal evidence that the PDGF B-type receptor mediates a motility response including actin reorganization and chemotaxis. Furthermore, the results establish a role for the receptor-associated protein-tyrosine kinase in the transduction of the chemotactic signal. Images PMID:2153283

  14. Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

    PubMed Central

    Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

    2015-01-01

    ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

  15. Slit2 involvement in glioma cell migration is mediated by Robo1 receptor.

    PubMed

    Mertsch, Sonja; Schmitz, Nicole; Jeibmann, Astrid; Geng, Jian-Guo; Paulus, Werner; Senner, Volker

    2008-03-01

    Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRT-PCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels. Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2 receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

  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. 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. Localization of endothelin ETA and ETB receptor-mediated constriction in the renal microcirculation of rats.

    PubMed Central

    Endlich, K; Hoffend, J; Steinhausen, M

    1996-01-01

    1. The aim of the study was to visualize endothelin-1 (ET-1)-mediated constriction in renal vessels of cortical and juxtamedullary glomeruli in the split hydronephrotic rat kidney in vivo and to functionally characterize the ET receptor subtypes involved. 2. ET-1 (10(-9) M) constricted preglomerular vessels (by 6-18%) and efferent arterioles (by 11-13%), and decreased glomerular blood flow (GBF, by 55%) of cortical and juxtamedullary glomeruli. 3. The ETA antagonist BQ-123 (10(-6) M), as well as the ETB antagonist BQ-788 (2 x 10(-7) M) and IRL 1038 (10(-6) M), shifted the concentration-response curve of GBF for ET-1 to the right by one order of magnitude. While BQ-123 antagonized ET-1 constriction only in preglomerular vessels, BQ-788 and IRL 1038 were effective both in preglomerular vessels and efferent arterioles. 4. The ETB agonist IRL 1620 (10(-8) M) reduced GBF by 50% and constricted efferent arterioles (by 20-33%) about two times more than preglomerular vessels (by 6-14%). 5. Our results suggest that in renal cortical and juxtamedullary vessels of rats, ET-1-induced preglomerular vasoconstriction is mediated by ETA and ETB receptors, while efferent vasoconstriction is predominantly mediated by ETB receptors, which might have important consequences for the regulation of glomerular filtration pressure by ET. PMID:8951723

  19. Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

    PubMed

    Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Zheng, Yao; Shan, Shuang; Li, Rui-Jun; Zhang, Yong-Jun; Guo, Yu-Yuan

    2016-07-01

    Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded "antennal IRs" and seven encoded "divergent IRs". Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception. PMID:27208597

  20. 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

  1. PreBotzinger complex neurokinin-1 receptor-expressing neurons mediate opioid-induced respiratory depression.

    PubMed

    Montandon, Gaspard; Qin, Wuxuan; Liu, Hattie; Ren, Jun; Greer, John J; Horner, Richard L

    2011-01-26

    The analgesic properties of the opium poppy Papever somniferum were first mentioned by Hippocrates around 400 BC, and opioid analgesics remain the mainstay of pain management today. These drugs can cause the serious side-effect of respiratory depression that can be lethal with overdose, however the critical brain sites and neurochemical identity of the neurons mediating this depression are unknown. By locally manipulating neurotransmission in the adult rat, we identify the critical site of the medulla, the preBötzinger complex, that mediates opioid-induced respiratory depression in vivo. Here we show that opioids at the preBötzinger complex cause respiratory depression or fatal apnea, with anesthesia and deep-sleep being particularly vulnerable states for opioid-induced respiratory depression. Importantly, we establish that the preBötzinger complex is fully responsible for respiratory rate suppression following systemic administration of opioid analgesics. The site in the medulla most sensitive to opioids corresponds to a region expressing neurokinin-1 receptors, and we show in rhythmically active brainstem section in vitro that neurokinin-1 receptor-expressing preBötzinger complex neurons are selectively inhibited by opioids. In summary, neurokinin-1 receptor-expressing preBötzinger complex neurons constitute the critical site mediating opioid-induced respiratory rate depression, and the key therapeutic target for its prevention or reversal.

  2. Inflammatory mediators and insulin resistance in obesity: role of nuclear receptor signaling in macrophages.

    PubMed

    Fuentes, Lucía; Roszer, Tamás; Ricote, Mercedes

    2010-01-01

    Visceral obesity is coupled to a general low-grade chronic inflammatory state characterized by macrophage activation and inflammatory cytokine production, leading to insulin resistance (IR). The balance between proinflammatory M1 and antiinflammatory M2 macrophage phenotypes within visceral adipose tissue appears to be crucially involved in the development of obesity-associated IR and consequent metabolic abnormalities. The ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs) have recently been implicated in the determination of the M1/M2 phenotype. Liver X receptors (LXRs), which form another subgroup of the nuclear receptor superfamily, are also important regulators of proinflammatory cytokine production in macrophages. Disregulation of macrophage-mediated inflammation by PPARs and LXRs therefore underlies the development of IR. This review summarizes the role of PPAR and LXR signaling in macrophages and current knowledge about the impact of these actions in the manifestation of IR and obesity comorbidities such as liver steatosis and diabetic osteopenia.

  3. 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.

  4. 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.

  5. Facilitation of AMPA receptor-mediated steady-state current by extrasynaptic NMDA receptors in supraoptic magnocellular neurosecretory cells

    PubMed Central

    Pai, Yoon Hyoung; Lim, Chae Seong; Park, Kyung-Ah; Cho, Hyun Sil; Lee, Gyu-Seung; Shin, Yong Sup; Kim, Hyun-Woo; Jeon, Byeong Hwa

    2016-01-01

    In addition to classical synaptic transmission, information is transmitted between cells via the activation of extrasynaptic receptors that generate persistent tonic current in the brain. While growing evidence supports the presence of tonic NMDA current (INMDA) generated by extrasynaptic NMDA receptors (eNMDARs), the functional significance of tonic INMDA in various brain regions remains poorly understood. Here, we demonstrate that activation of eNMDARs that generate INMDA facilitates the α-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor (AMPAR)-mediated steady-state current in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs). In low-Mg2+ artificial cerebrospinal fluid (aCSF), glutamate induced an inward shift in Iholding (IGLU) at a holding potential (Vholding) of –70 mV which was partly blocked by an AMPAR antagonist, NBQX. NBQX-sensitive IGLU was observed even in normal aCSF at Vholding of –40 mV or –20 mV. IGLU was completely abolished by pretreatment with an NMDAR blocker, AP5, under all tested conditions. AMPA induced a reproducible inward shift in Iholding (IAMPA) in SON MNCs. Pretreatment with AP5 attenuated IAMPA amplitudes to ~60% of the control levels in low-Mg2+ aCSF, but not in normal aCSF at Vholding of –70 mV. IAMPA attenuation by AP5 was also prominent in normal aCSF at depolarized holding potentials. Memantine, an eNMDAR blocker, mimicked the AP5-induced IAMPA attenuation in SON MNCs. Finally, chronic dehydration did not affect IAMPA attenuation by AP5 in the neurons. These results suggest that tonic INMDA, mediated by eNMDAR, facilitates AMPAR function, changing the postsynaptic response to its agonists in normal and osmotically challenged SON MNCs. PMID:27382359

  6. Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway.

    PubMed

    Reeves, Patrick M; Kang, Yuan-Lin; Kirchhausen, Tom

    2016-01-01

    The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin 1 and β-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s.

  7. Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

    SciTech Connect

    Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao

    2008-06-20

    We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor {gamma}-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

  8. Mechanism of HSV infection through soluble adapter-mediated virus bridging to the EGF receptor

    SciTech Connect

    Nakano, Kenji; Kobayashi, Masatoshi; Nakamura, Kei-ichiro; Nakanishi, Takeshi; Asano, Ryutaro; Kumagai, Izumi; Tahara, Hideaki; Kuwano, Michihiko; Cohen, Justus B.; Glorioso, Joseph C.

    2011-04-25

    Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.

  9. IL-6 Overexpression in ERG-Positive Prostate Cancer Is Mediated by Prostaglandin Receptor EP2.

    PubMed

    Merz, Constanze; von Mässenhausen, Anne; Queisser, Angela; Vogel, Wenzel; Andrén, Ove; Kirfel, Jutta; Duensing, Stefan; Perner, Sven; Nowak, Michael

    2016-04-01

    Prostate cancer is the most diagnosed cancer in men and multiple risk factors and genetic alterations have been described. The TMPRSS2-ERG fusion event and the overexpression of the transcription factor ERG are present in approximately 50% of all prostate cancer patients, however, the clinical outcome is still controversial. Prostate tumors produce various soluble factors, including the pleiotropic cytokine IL-6, regulating cellular processes such as proliferation and metastatic segregation. Here, we used prostatectomy samples in a tissue microarray format and analyzed the co-expression and the clinicopathologic data of ERG and IL-6 using immunohistochemical double staining and correlated the read-out with clinicopathologic data. Expression of ERG and IL-6 correlated strongly in prostate tissue samples. Forced expression of ERG in prostate tumor cell lines resulted in significantly increased secretion of IL-6, whereas the down-regulation of ERG decreased IL-6 secretion. By dissecting the underlying mechanism in prostate tumor cell lines we show the ERG-mediated up-regulation of the prostanoid receptors EP2 and EP3. The prostanoid receptor EP2 was overexpressed in human prostate cancer tissue. Furthermore, the proliferation rate and IL-6 secretion in DU145 cells was reduced after treatment with EP2-receptor antagonist. Collectively, our study shows that the expression of ERG in prostate cancer is linked to the expression of IL-6 mediated by the prostanoid receptor EP2.

  10. 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

  11. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  12. 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

  13. Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Scrogin, K. E.; Johnson, A. K.; Schmid, H. A.

    1998-01-01

    The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

  14. 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.

  15. Angiotensin II AT2 receptors regulate NGF-mediated neurite outgrowth via the NO-cGMP pathway.

    PubMed

    Hashikawa-Hobara, Narumi; Hashikawa, Naoya

    2016-09-16

    We investigated whether Angiotensin II type 2 (AT2) receptor activation was involved in NGF-induced nerve regeneration. NGF-mediated neurite outgrowth in cultured dorsal root ganglia (DRG) cells was significantly inhibited by AT2 receptor antagonist (PD123,319) treatment. AT2 receptor knockdown also inhibited NGF-mediated neurite outgrowth. To determine the mechanisms, we analyzed the NO-cGMP pathway. The cGMP analog increased NGF-mediated nerve elongation, which inhibited by PD123,319. Furthermore, soluble guanylate cyclase expression was significantly less in NGF and PD123,319 treatment DRG than in NGF treatment alone. These results suggest that NGF-mediated neurite outgrowth is suppressed by AT2 receptor signaling via the NO-cGMP-PKG pathway. PMID:27524238

  16. Liver X receptor agonists decrease ENaC-mediated sodium transport in collecting duct cells.

    PubMed

    Soodvilai, Sunhapas; Jia, Zhanjun; Fongsupa, Somsak; Chatsudthipong, Varanuj; Yang, Tianxin

    2012-12-15

    Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol, fatty acid, and glucose metabolism in various tissues. However, the renal action of LXRs is not well understood. Here we investigated the effects of LXR-activating ligands on modulation of epithelial sodium channel (ENaC)-mediated sodium transport in collecting duct cells. Exposure of the M1 cells to the synthetic LXR agonists T0901317 and GW3965 or the natural ligand 22R-hydroxycholesterol for 24 h decreased amiloride-sensitive sodium transport, corresponding with an increase of transepithelial resistance. The inhibition of amiloride-sensitive sodium transport after incubation with T0901317 or GW3965 was not mediated by a reduction of Na(+)/K(+)-ATPase-mediated basolateral sodium transport. On the other hand, T0901317 and GW3965 decreased mRNA abundance and membrane expression of ENaC. Preincubation the monolayer with GW3965 attenuated aldosterone-induced stimulation sodium transport. In primary cultures of collecting duct cells, T0901317 and GW3965 similarly inhibited ENaC transport function as in M1 cells. This is the first evidence showing LXR-activating ligands modulate ENaC-mediated sodium transport in collecting duct cells. These results suggest that LXRs may represent a novel therapeutic target for treatment of conditions with dysregulation of ENaC such as hypertension.

  17. Proteinase-activated receptors 1 and 2 mediate contraction of human oesophageal muscularis mucosae.

    PubMed

    Chang, B-S; Chang, J-C; Huang, S-C

    2010-01-01

    Proteinase-activated receptors 1 and 2 mediate contraction of the human gallbladder. In the present study, we investigated effects mediated by proteinase-activated receptors (PARs) in the human oesophagus by measuring contraction of muscularis mucosae strips isolated from the human oesophagus. Both PAR(1) agonists (thrombin, SFLLRN-NH(2) and TFLLR-NH(2)) and PAR(2) agonists (trypsin, 2-furoyl-LIGRLO-NH(2) and SLIGKV-NH(2)) caused concentration-dependent contraction. In contrast, PAR(1) and PAR(2) control peptides did not cause contraction. The existence of PAR(1) and PAR(2) in the human oesophageal muscularis mucosae was confirmed by immunohistochemistry and reverse transcription-polymerase chain reaction. On the other hand, PAR(4) agonists, GYPGKF-NH(2), GYPGQV-NH(2) and AYPGKF-NH(2), did not cause contraction or relaxation in resting or carbachol-contracted muscularis mucosae strips, suggesting that PAR(4) is not involved in human oesophageal motility. The contractile responses to SFLLRN-NH(2) and trypsin in the human oesophagus were insensitive to atropine and tetrodotoxin, indicating that the contractile response was not neurally mediated. Taken together, these results demonstrate that PAR(1) and PAR(2) but not PAR(4) mediate contraction in human oesophageal muscularis mucosae. PAR(1) and PAR(2) may influence human oesophageal motility. PMID:19694963

  18. Promiscuous Dimerization of the Growth Hormone Secretagogue Receptor (GHS-R1a) Attenuates Ghrelin-mediated Signaling*

    PubMed Central

    Schellekens, Harriët; van Oeffelen, Wesley E. P. A.; Dinan, Timothy G.; Cryan, John F.

    2013-01-01

    G protein-coupled receptors (GPCRs), such as the ghrelin receptor (GHS-R1a), the melanocortin 3 receptor (MC3), and the serotonin 2C receptor (5-HT2C), are well known for their key role in the homeostatic control of food intake and energy balance. Ghrelin is the only known gut peptide exerting an orexigenic effect and has thus received much attention as an anti-obesity drug target. In addition, recent data have revealed a critical role for ghrelin in dopaminergic mesolimbic circuits involved in food reward signaling. This study investigates the downstream signaling consequences and ligand-mediated co-internalization following heterodimerization of the GHS-R1a receptor with the dopamine 1 receptor, as well as that of the GHS-R1a-MC3 heterodimer. In addition, a novel heterodimer between the GHS-R1a receptor and the 5-HT2C receptor was identified. Interestingly, dimerization of the GHS-R1a receptor with the unedited 5-HT2C-INI receptor, but not with the partially edited 5-HT2C-VSV isoform, significantly reduced GHS-R1a agonist-mediated calcium influx, which was completely restored following pharmacological blockade of the 5-HT2C receptor. These results combined suggest a potential novel mechanism for fine-tuning GHS-R1a receptor-mediated activity via promiscuous dimerization of the GHS-R1a receptor with other G protein-coupled receptors involved in appetite regulation and food reward. These findings may uncover novel mechanisms of significant relevance for the future pharmacological targeting of the GHS-R1a receptor in the homeostatic regulation of energy balance and in hedonic appetite signaling, both of which play a significant role in the development of obesity. PMID:23161547

  19. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  20. 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

  1. 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

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

    PubMed

    Zanchet, Eliane Maria; Cury, Yara

    2003-04-25

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

  3. Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors.

    PubMed

    Scislo, Tadeusz J; O'Leary, Donal S

    2002-10-01

    Selective activation of adenosine A(1) and A(2a) receptors in the subpostremal nucleus tractus solitarius (NTS) increases and decreases mean arterial pressure (MAP), respectively, and decreases heart rate (HR). We have previously shown that the decreases in MAP evoked by NTS A(2a) receptor stimulation were accompanied with differential sympathetic responses in renal (RSNA), lumbar (LSNA), and preganglionic adrenal sympathetic nerve activity (pre-ASNA). Therefore, now we investigated whether stimulation of NTS A(1) receptors via unilateral microinjection of N(6)-cyclopentyladenosine (CPA) elicits differential activation of the same sympathetic outputs in alpha-chloralose-urethane-anesthetized male Sprague-Dawley rats. CPA (0.33-330.0 pmol in 50 nl) evoked dose-dependent increases in MAP, variable decreases in HR, and differential increases in all recorded sympathetic outputs: upward arrow pre-ASNA > upward arrow RSNA > or = upward arrow LSNA. Sinoaortic denervation + vagotomy abolished the MAP and LSNA responses, reversed the normal increases in RSNA into decreases, and significantly attenuated increases in pre-ASNA. NTS ionotropic glutamatergic receptor blockade with kynurenate sodium (4.4 nmol/100 nl) reversed the responses in MAP, LSNA, and RSNA and attenuated the responses in pre-ASNA. We conclude that afferent inputs and intact glutamatergic transmission in the NTS are necessary to mediate the pressor and differential sympathoactivatory responses to stimulation of NTS A(1) receptors.

  4. Alpha adrenergic receptor mediation of cardiovascular and metabolic responses to alcohol

    SciTech Connect

    Brackett, D.J.; Gauvin, D.V.; Lerner, M.R.; Holloway, F.H.; Wilson, M.F. Veterans Affairs Medical Center, Oklahoma City, OK )

    1992-02-26

    The role of alpha adrenergic receptors in acute cardiovascular and metabolic responses to alcohol (ETOH) have not been clearly defined. In this study two groups of male Sprague-Dawley rats were given intravenous phentolamine mesylate or saline prior to intragastric alcohol to blockade of alpha receptors during alcohol intoxication in conscious rats. ETOH alone evoked an increase in systemic vascular resistance (SVR), heart rate (HR), and blood glucose concentrations (G) and a decrease in mean arterial pressure (MAP), cardiac output (CO), central venous pressure (CVP), respiration rate (RR) and cardiac stroke volume (SV). Blood alcohol concentration (BAC) peaked at 30 min and remained elevated for the four hrs of monitoring. Phentolamine pretreatment produced a decrease in MAP and SV and an increase in HR. However, antagonism of the alpha receptor blocked the decrease in CO and the increase in SVR and G. The decrease in CVP was unaffected. Surprisingly, the early rise and peak in BAC in the phentolamine treated group was attenuated, but was the same as the untreated group during the final 3 hrs. These data suggest that alpha receptors are significant mediators of cardiovascular and glucoregulatory responses elicited by alcohol. Furthermore, alpha receptor blockade appears to effect the absorption and/or distribution of intragastrically administered alcohol.

  5. Resolvin E1 and chemokine-like receptor 1 mediate bone preservation.

    PubMed

    Gao, Li; Faibish, Dan; Fredman, Gabrielle; Herrera, Bruno S; Chiang, Nan; Serhan, Charles N; Van Dyke, Thomas E; Gyurko, Robert

    2013-01-15

    The polyunsaturated ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions. PMID:23241890

  6. Receptor subtypes mediating depressor responses to microinjections of nicotine into medial NTS of the rat.

    PubMed

    Dhar, S; Nagy, F; McIntosh, J M; Sapru, H N

    2000-07-01

    Microinjections (50 nl) of nicotine (0.01-10 microM) into the nucleus of the solitary tract (NTS) of adult, urethan-anesthetized, artificially ventilated, male Wistar rats, elicited decreases in blood pressure and heart rate. Prior microinjections of alpha-bungarotoxin (alpha-BT) and alpha-conotoxin ImI (specific toxins for nicotinic receptors containing alpha7 subunits) elicited a 20-38% reduction in nicotine responses. Similarly, prior microinjections of hexamethonium, mecamylamine, and alpha-conotoxin AuIB (specific blockers or toxin for nicotinic receptors containing alpha3beta4 subunits) elicited a 47-79% reduction in nicotine responses. Nicotine responses were completely blocked by prior sequential microinjections of alpha-BT and mecamylamine into the NTS. Complete blockade of excitatory amino acid receptors (EAARs) in the NTS did not attenuate the responses to nicotine. It was concluded that 1) the predominant type of nicotinic receptor in the NTS contains alpha3beta4 subunits, 2) a smaller proportion contains alpha7 subunits, 3) the presynaptic nicotinic receptors in the NTS do not contribute to nicotine-induced responses, and 4) EAARs in the NTS are not involved in mediating responses to nicotine.

  7. 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.

  8. The nuclear architectural protein HMGA1a triggers receptor-mediated endocytosis.

    PubMed

    Wu, Wuwei; Wan, Wei; Li, Alexander D Q

    2009-11-01

    High mobility group proteins A (HMGA), nuclear architectural factors, locate in the cell nuclei and mostly execute gene-regulation function. However, our results reveal that a HMGA member (HMGA1a) has a unique plasma membrane receptor; this receptor specifically binds to HMGA-decorated species, effectively mediates endocytosis, and internalizes extracellular HMGA-functionalized cargoes. Indeed, dyes or nanoparticles labeled with HMGA1a protein readily enter Hela cells. Using a stratagem chemical cross-linker, we covalently bonded the HMGA receptor to the HMGA1a-GFP fusion protein, thus capturing the plasma membrane receptor. Subsequent Western blots and SDS-PAGE gel revealed that the HMGA receptor is a 26-kDa protein. Confocal live-cell microscopic imaging was used to monitor the whole endocytic process, in which the internalized HMGA1a-decorated species are transported by motor proteins on microtubules and eventually arrive at the late endosomes/lysosomes. Cell viability assays also suggested that extracellular HMGA1a protein directly influences the survival ability of Hela cells in a dose-dependent manner, implying versatility of HMGA1a protein and its potent role to suppress cancer cell survivability and to regulate growth. PMID:19739099

  9. Neuronal protease-activated receptor 1 drives synaptic retrograde signaling mediated by the endocannabinoid 2-arachidonoylglycerol.

    PubMed

    Hashimotodani, Yuki; Ohno-Shosaku, Takako; Yamazaki, Maya; Sakimura, Kenji; Kano, Masanobu

    2011-02-23

    Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB(1) receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid 2-arachidonoylglycerol (2-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca(2+) elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB(1) receptors and suppresses transmitter release at hippocampal inhibitory synapses.

  10. 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.

  11. Involvement of NMDA receptors in nicotine-mediated central control of hypotensive effects.

    PubMed

    Hong, Ling-Zong; Cheng, Pei-Wen; Cheng, Wen-Han; Chen, Siang-Ru; Wang, Ling-Lin; Tseng, Ching-Jiunn

    2012-10-31

    It is known that enrichment of glutamatergic transmission in the nucleus tractus solitarii (NTS) plays an important role in central cardiovascular regulation. Our previous study demonstrated that nicotine decreased blood pressure and heart rate in the NTS probably acting via the nicotinic acetylcholine receptors (nAChRs)-Ca²⁺-calmodulin-eNOS-NO signaling pathway. The possible relationship between glutamate and nicotine in the NTS for cardiovascular regulation is poorly understood. This study investigated the involvement of glutamate receptors in the cardiovascular effects of nicotine in the NTS. Nicotine (a non-selective nAChRs agonist), MK801 (a non-competitive NMDA receptor antagonist), APV (a competitive NMDA receptor antagonist), or NBQX (a selective AMPA receptor antagonist) was microinjected into the NTS of anesthetized Wistar-Kyoto rats. Microinjection of nicotine (1.5 pmol) into the NTS produced decreases in blood pressure and heart rate. The hypotensive and bradycardic effects of nicotine were abolished by prior administration of MK801 (1 nmol) and APV (10 nmol), but was completely restored after 60 min of recovery. In contrast, prior administration of NBQX (10 pmol) into the NTS did not alter the cardiovascular effects of nicotine. The nitrate (served as total NO) production in response to nicotine microinjection into the NTS was suppressed by prior administration of APV. These results suggest that the hypotensive and bradycardic effects of nicotine in the NTS might be mediated through NMDA receptors, and that the nAChRs-NMDA receptor-NO pathway could be involved.

  12. 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

  13. Real-Time Imaging of Discrete Exocytic Events Mediating Surface Delivery of AMPA Receptors

    PubMed Central

    Yudowski, Guillermo A.; Puthenveedu, Manojkumar A.; Leonoudakis, Dmitri; Panicker, Sandip; Thorn, Kurt S.; Beattie, Eric C.; von Zastrow, Mark

    2011-01-01

    We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using single-molecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. “Transient” events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within ~1 s) increase in surface AMPAR fluorescence extending locally (2–5µm) from the site of exocytosis. “Persistent” events, from which inserted AMPARs dispersed slowly (typically over 5–10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activity-dependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses. PMID:17928453

  14. 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.

  15. 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).

  16. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro.

    PubMed Central

    Fuki, I V; Kuhn, K M; Lomazov, I R; Rothman, V L; Tuszynski, G P; Iozzo, R V; Swenson, T L; Fisher, E A; Williams, K J

    1997-01-01

    Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo. PMID:9294130

  17. Fenugreek induced apoptosis in breast cancer MCF-7 cells mediated independently by fas receptor change.

    PubMed

    Alshatwi, Ali Abdullah; Shafi, Gowhar; Hasan, Tarique Noorul; Syed, Naveed Ahmed; Khoja, Kholoud Khalid

    2013-01-01

    Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently. PMID:24289578

  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. Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

    PubMed

    Roth, Lise; Prahst, Claudia; Ruckdeschel, Tina; Savant, Soniya; Weström, Simone; Fantin, Alessandro; Riedel, Maria; Héroult, Mélanie; Ruhrberg, Christiana; Augustin, Hellmut G

    2016-04-26

    Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

  20. Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling

    SciTech Connect

    Kakihana, Kazuhiko; Yamamoto, Masahide; Iiyama, Mitsuko; Miura, Osamu . E-mail: miura.hema@tmd.ac.jp

    2005-09-23

    Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca{sup 2+} level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca{sup 2+} receptor calmodulin (CaM) binds EpoR in a Ca{sup 2+}-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258-312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

  1. 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.

  2. Hydroxycarboxylic acid receptor 2 mediates dimethyl fumarate’s protective effect in EAE

    PubMed Central

    Chen, Hui; Assmann, Julian C.; Krenz, Antje; Rahman, Mahbubur; Grimm, Myriam; Karsten, Christian M.; Köhl, Jörg; Offermanns, Stefan; Wettschureck, Nina; Schwaninger, Markus

    2014-01-01

    Taken orally, the drug dimethyl fumarate (DMF) has been shown to improve functional outcomes for patients with MS; however, it is unclear how DMF mediates a protective effect. DMF and, more so, its active metabolite, monomethyl fumarate, are known agonists of the hydroxycarboxylic acid receptor 2 (HCA2), a G protein–coupled membrane receptor. Here, we evaluated the contribution of HCA2 in mediating the protective effect afforded by DMF in EAE, a mouse model of MS. DMF treatment reduced neurological deficit, immune cell infiltration, and demyelination of the spinal cords in wild-type mice, but not in Hca2–/– mice, indicating that HCA2 is required for the therapeutic effect of DMF. In particular, DMF decreased the number of infiltrating neutrophils in a HCA2-dependent manner, likely by interfering with neutrophil adhesion to endothelial cells and chemotaxis. Together, our data indicate that HCA2 mediates the therapeutic effects of DMF in EAE. Furthermore, identification of HCA2 as a molecular target may help to optimize MS therapy. PMID:24691444

  3. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

    PubMed

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-07-01

    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

  4. Dual effects of nicotine on dopamine neurons mediated by different nicotinic receptor subtypes.

    PubMed

    Schilström, Björn; Rawal, Nina; Mameli-Engvall, Monica; Nomikos, George G; Svensson, Torgny H

    2003-03-01

    Burst firing of dopaminergic neurons has been found to represent a particularly effective means of increasing dopamine release in terminal areas as well as activating immediate early genes in dopaminoceptive cells. Spontaneous burst firing is largely controlled by the level of activation of NMDA receptors in the ventral tegmental area (VTA) as a consequence of glutamate released from afferents arising mainly in the prefrontal cortex. Nicotine has been found to effectively increase burst firing of dopaminergic cells. This effect of nicotine may be due to an alpha 7 nicotinic receptor-mediated presynaptic facilitation of glutamate release in the VTA. By the use of in-vivo single-cell recordings and immunohistochemistry we here evaluated the role of alpha 7 nicotinic receptors in nicotine-induced burst firing of dopamine cells in the VTA and the subsequent activation of immediate early genes in dopaminoceptive target areas. Nicotine (0.5 mg/kg s.c.) was found to increase firing rate and burst firing of dopaminergic neurons. In the presence of methyllycaconitine (MLA, 6.0 mg/kg i.p.) nicotine only increased firing rate. Moreover, in the presence of dihydro-beta-erythroidine (DH beta E, 1.0 mg/kg i.p.), an antagonist at non-alpha 7 nicotinic receptors, nicotine produced an increase in burst firing without increasing the firing rate. Nicotine also increased Fos-like immunoreactivity in dopamine target areas, an effect that was antagonized with MLA but not with DH beta E. Our data suggest that nicotine's augmenting effect on burst firing is, indeed, due to stimulation of alpha 7 nicotinic receptors whereas other nicotinic receptors seem to induce an increase in firing frequency.

  5. Estrogen and Estrogen Receptor-α-Mediated Transrepression of Bile Salt Export Pump

    PubMed Central

    Chen, Yuan; Vasilenko, Alex; Song, Xiulong; Valanejad, Leila; Verma, Ruchi; You, Sangmin; Yan, Bingfang; Shiffka, Stephanie; Hargreaves, Leeza; Nadolny, Christina

    2015-01-01

    Among diseases unique to pregnancy, intrahepatic cholestasis of pregnancy is the most prevalent disorder with elevated serum bile acid levels. We have previously shown that estrogen 17β-estradiol (E2) transrepresses bile salt export pump (BSEP) through an interaction between estrogen receptor (ER)-α and farnesoid X receptor (FXR) and transrepression of BSEP by E2/ERα is an etiological contributing factor to intrahepatic cholestasis of pregnancy. Currently the mechanistic insights into such transrepression are not fully understood. In this study, the dynamics of coregulator recruitment to BSEP promoter after FXR activation and E2 treatment were established with quantitative chromatin immunoprecipitation assays. Coactivator peroxisome proliferator-activated receptor-γ coactivator-1 was predominantly recruited to the BSEP promoter upon FXR activation, and its recruitment was decreased by E2 treatment. Meanwhile, recruitment of nuclear receptor corepressor was markedly increased upon E2 treatment. Functional evaluation of ERα and ERβ chimeras revealed that domains AC of ERα are the determinants for ERα-specific transrepression on BSEP. Further studies with various truncated ERα proteins identified the domains in ERα responsible for ligand-dependent and ligand-independent transrepression. Truncated ERα-AD exhibited potent ligand-independent transrepressive activity, whereas ERα-CF was fully capable of transrepressing BSEP ligand dependently in vitro in Huh 7 cells and in vivo in mice. Both ERα-AD and ERα-CF proteins were associated with FXR in the coimmunoprecipitation assays. In conclusion, E2 repressed BSEP expression through diminishing peroxisome proliferator-activated receptor-γ coactivator-1 recruitment with a concurrent increase in nuclear receptor corepressor recruitment to the BSEP promoter. Domains AD and CF in ERα mediated ligand-independent and ligand-dependent transrepression on BSEP, respectively, through interacting with FXR. PMID:25675114

  6. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells

    PubMed Central

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-01

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  7. 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

  8. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    NASA Astrophysics Data System (ADS)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  9. Identification of the ETA receptor subtype that mediates endothelin induced autocrine proliferation of normal human keratinocytes.

    PubMed

    Bagnato, A; Venuti, A; Di Castro, V; Marcante, M L

    1995-04-01

    Endothelin-1 has a wide range of pharmacological effects in various tissues and acts as autocrine/paracrine factor. The potential of ET-1 to function as an autocrine growth factor was evaluated in normal human keratinocytes. Radioligand binding studies showed that 125I-ET-1 bound to a single class of high-affinity-binding sites on the surface of the cells. The dissociation constant was 0.045 nM with receptor numbers of 1700 sites/cell. Treatment with serum caused increases in expression of binding sites (3500 sites/cell), with no change in binding affinity. ET-1 stimulated thymidine incorporation in these cells that expressed ET receptors. An ET antagonist selective for the ETA receptor subtype (BQ 123) inhibited DNA synthesis stimulated by ET-1 and reduced the basal growth rate of unstimulated cells. These data suggest that the ET-1 induced DNA synthesis is mediated by ETA receptor subtype and that endogenously produced ET-1 promotes the autocrine proliferation of keratinocytes.

  10. Tissue kallikrein mediates neurite outgrowth through epidermal growth factor receptor and flotillin-2 pathway in vitro.

    PubMed

    Lu, Zhengyu; Cui, Mei; Zhao, Hong; Wang, Tao; Shen, Yan; Dong, Qiang

    2014-02-01

    Tissue kallikrein (TK) was previously shown to take most of its biological effects through bradykinin receptors. In this study, we assumed that TK mediated neurite outgrowth was independent of bradykinin receptors. To test the hypothesis, we investigated TK-induced neurite outgrowth and its signaling mechanisms in cultured primary neurons and human SH-SY5Y cells. We found that TK stimulation could increase the number of processes and mean process length of primary neurons, which were blocked by epidermal growth factor receptor (EGFR) inhibitor or down-regulation, small interfering RNA for flotillin-2 and extracellular signal-regulated kinase (ERK) 1/2 inhibitor. Moreover, TK-induced neurite outgrowth was associated with EGFR and ERK1/2 activation, which were inhibited by EGFR antagonist or RNA interference and flotillin-2 knockdown. Interestingly, inhibition of bradykinin receptors had no significant effects on EGFR and ERK1/2 phosphorylation. In the present research, our data also suggested that EGFR and flotillin-2 formed constitutive complex that translocated to around the nuclei in the TK stimulation. In sum, our findings provided evidence that TK could promote neurite outgrowth via EGFR, flotillin-2 and ERK1/2 signaling pathway in vitro. PMID:24211626

  11. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    PubMed Central

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  12. 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

  13. 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

  14. Agonist mediated conformational changes of solubilized calf forebrain muscarinic acetylcholine receptors.

    PubMed

    Vanderheyden, P; Andre, C; de Backer, J P; Vauquelin, G

    1984-10-01

    Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations with respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85 +/- 5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation. PMID:6487351

  15. Adrenergic and serotoninergic receptors mediate the immunological activation of corticosterone secretion in male rats.

    PubMed

    Guo, A L; Petraglia, F; Criscuolo, M; Ficarra, G; Salvestroni, C; Nappi, R E; Trentini, G P; Genazzani, A R

    1996-06-01

    In order to elucidate the mechanism of action of immune agents on corticosterone secretion, the present study evaluated the possible involvement of some neuronal pathways (serotoninergic, noradrenergic/adrenergic) in the lipopolysaccharide (LPS)-induced corticosterone release in male rats. Serotoninergic antagonists, mianserin (5-HT2C receptor blocker) or pindolol (5HT1A receptor blocker) or noradrenergic/adrenergic antagonists, prazosin (alpha 1-adrenoceptor blocker) or propranolol (beta-adrenoceptor blocker), were intraperitoneally (i.p.) injected before (5 min) the administration of LPS. In each experiment a group of rats i.p. injected with vehicle served as controls. Animals were sacrificed by decapitation 90 min after administration of LPS and trunk blood was collected for corticosterone radioimmunoassay. Results showed that pretreatment with mianserin, but not with pindolol, significantly reduced plasma corticosterone levels following administration of LPS (p < 0.05); prazosin attenuated the plasma corticosterone response to LPS (p < 0.05), while propranolol did not induce significant change. The present study indicated that serotoninergic and noradrenergic/adrenergic pathways are involved in the immunoneuroendocrine modulation of hypothalamus-pituitary-adrenal function in rats. In particular, it is probably mediated by the activation of 5-HT2C receptors and of alpha 1-adrenoceptors, while type 1A serotonin receptors or beta-adrenoceptors do not seem to be involved in such a phenomenon.

  16. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    PubMed

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  17. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor.

    PubMed

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-20

    Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A2A adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from ∼O(10(2)) ps to ∼O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (∼O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs. PMID:27653477

  18. Augmentation of glycine receptor alpha3 currents suggests a mechanism for glucose-mediated analgesia.

    PubMed

    Breitinger, Ulrike; Breitinger, Hans-Georg

    2016-01-26

    The inhibitory glycine receptor (GlyR) mediates rapid synaptic inhibition in the mammalian central nervous system. Recently, glucose was identified as a positive modulator of α1 GlyRs. Here, recombinant human α3GlyRs with and without glucose treatment were studied using patch clamp methods. Similar to α1GlyRs, receptor variants α3L and α3K were potentiated by sugar. Glucose treatment reduced EC50 values of GlyR α3L and α3K by a factor of 4.5 and 3.3, respectively, without affecting maximum currents or desensitization. The high-activity mutant α3L(P185L) was not further potentiated by glucose. Potentiation of glycinergic signalling may underlie some of the analgetic effects of glucose. PMID:26656729

  19. The complexity of signalling mediated by the glucagon-like peptide-1 receptor.

    PubMed

    Fletcher, Madeleine M; Halls, Michelle L; Christopoulos, Arthur; Sexton, Patrick M; Wootten, Denise

    2016-04-15

    The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR that is a major therapeutic target for the treatment of type 2 diabetes. The receptor is activated by the incretin peptide GLP-1 promoting a broad range of physiological effects including glucose-dependent insulin secretion and biosynthesis, improved insulin sensitivity of peripheral tissues, preservation of β-cell mass and weight loss, all of which are beneficial in the treatment of type 2 diabetes. Despite this, existing knowledge surrounding the underlying signalling mechanisms responsible for the physiological actions downstream of GLP-1R activation is limited. Here, we review the current understanding around GLP-1R-mediated signalling, in particular highlighting recent contributions to the field on biased agonism, the spatial and temporal aspects for the control of signalling and how these concepts may influence future drug development. PMID:27068973

  20. Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics

    PubMed Central

    Rust, Marco B; Gurniak, Christine B; Renner, Marianne; Vara, Hugo; Morando, Laura; Görlich, Andreas; Sassoè-Pognetto, Marco; Banchaabouchi, Mumna Al; Giustetto, Maurizio; Triller, Antoine; Choquet, Daniel; Witke, Walter

    2010-01-01

    Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability. PMID:20407421

  1. 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.

  2. Presynaptic long-term depression mediated by Gi/o-coupled receptors

    PubMed Central

    Atwood, Brady K.; Lovinger, David M.; Mathur, Brian N.

    2014-01-01

    Long-term depression (LTD) of the efficacy of synaptic transmission is now recognized as an important mechanism for regulation of information storage and control of actions, as well as synapse, neuron, and circuit development. Studies of LTD mechanisms have focused mainly on postsynaptic AMPA receptor trafficking. However, the focus has now expanded to include presynaptically expressed plasticity; the predominant form being initiated by presynaptically expressed Gi/o-coupled metabotropic receptor (Gi/o-GPCR) activation. Several forms of LTD involving activation of different presynaptic Gi/o-GPCRs as a “common pathway” are described. Here, we review the literature on presynaptic Gi/o-GPCR-mediated LTD, discuss known mechanisms, gaps in our knowledge, and evaluate if all Gi/o-GPCR are capable of inducing presynaptic LTD. PMID:25160683

  3. Augmentation of glycine receptor alpha3 currents suggests a mechanism for glucose-mediated analgesia.

    PubMed

    Breitinger, Ulrike; Breitinger, Hans-Georg

    2016-01-26

    The inhibitory glycine receptor (GlyR) mediates rapid synaptic inhibition in the mammalian central nervous system. Recently, glucose was identified as a positive modulator of α1 GlyRs. Here, recombinant human α3GlyRs with and without glucose treatment were studied using patch clamp methods. Similar to α1GlyRs, receptor variants α3L and α3K were potentiated by sugar. Glucose treatment reduced EC50 values of GlyR α3L and α3K by a factor of 4.5 and 3.3, respectively, without affecting maximum currents or desensitization. The high-activity mutant α3L(P185L) was not further potentiated by glucose. Potentiation of glycinergic signalling may underlie some of the analgetic effects of glucose.

  4. 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.

  5. Truncated SRA RNA derivatives inhibit estrogen receptor-α-mediated transcription.

    PubMed

    Jung, Euihan; Jang, Seonghui; Lee, Jungmin; Kim, Youngmi; Shin, Heegwon; Park, Hee-Sung; Lee, Younghoon

    2016-10-01

    The steroid receptor RNA activator (SRA) is a long non-coding RNA (lncRNA) that acts as a putative coactivator for steroid receptor-mediated transcription. A recent study showed that SRA RNA can be structurally dissected into four domains comprising various secondary structures, but the contribution of each domain to the coactivation ability of SRA RNA was previously unknown. Here, we assessed the functional contributions of the various domains of SRA. We examined the effects of each domain on the coactivation of estrogen receptor-α (ERα)-mediated transcription of a luciferase reporter gene in HeLa cells. Then the detailed domain analysis was focused on domain III (D3) not only with the reporter gene in HeLa cells, but also with ERα-responsive genes in MCF7 breast cancer cells. Domain deletion analysis showed that the deletion of any domain decreased the luciferase activity, and that deletion of D3 caused the largest decrease. This D3 deletion effect was not recovered by co-expression of D3 alone; moreover, the expression of D3 fragments (particularly helices H15-H18, which are highly conserved across vertebrates) inhibited luciferase expression in HeLa cells. Moreover, a fragment containing helices H15-H18 reduced ERα-responsive gene expression in MCF7 breast cancer cells. Our findings indicate that D3 inhibited ERα-mediated transcription of a reporter gene in HeLa cells and that helices H15-H18, as a core element responsible for the D3-driven inhibition, reduced expression of ERα-responsive genes in breast cancer cells.

  6. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice.

    PubMed

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J

    2013-05-01

    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

  7. 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

  8. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures. PMID:26874070

  9. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  10. Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3

    PubMed Central

    Gui, Chunshan; Miao, Yi; Thompson, Lucas; Wahlgren, Bret; Mock, Melissa; Stieger, Bruno; Hagenbuch, Bruno

    2008-01-01

    The pregnane X receptor is a ligand-activated transcription factor that is abundantly expressed in hepatocytes. Numerous drugs are pregnane X receptor ligands. To bind to their receptor they must cross the sinusoidal membrane. Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are polyspecific transporters expressed at the sinusoidal membrane of human hepatocytes. They mediate transport of a variety of drugs including the pregnane X receptor ligands rifampicin and dexamethasone. To test whether additional pregnane X receptor ligands interact with OATP1B1- and 1B3-mediated transport, we developed Chinese Hamster Ovary (CHO) cell lines stably expressing OATP1B1 or 1B3 at high levels. OATP1B1- and 1B3-mediated estradiol-17β-glucuronide uptake was inhibited by several pregnane X receptor ligands in a concentration dependent way. IC50 values for rifampicin, paclitaxel, mifepristone, and troglitazone were within their respective pharmacological free plasma concentrations. Kinetic analysis revealed that clotrimazole inhibits OATP1B1-mediated estradiol-17β-glucuronide transport with a Ki of 7.7 ± 0.3 μM in a competitive way. However, uptake of OATP1B3-mediated estradiol-17β-glucuronide was stimulated and this stimulation was due to an increased apparent affinity. Transport of estrone-3-sulfate was hardly affected while all other substrates tested were inhibited. Additional azoles like fluconazole, ketoconazole and miconazole did not stimulate OATP1B3-mediated estradiol-17β-glucuronide transport. In summary, these results demonstrate that pregnane X receptor ligands, by inhibiting or stimulating OATP-mediated uptake, can lead to drug-drug interactions at the transporter level. PMID:18321482

  11. Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

    PubMed

    van der Putten, Céline; Zuiderwijk-Sick, Ella A; van Straalen, Linda; de Geus, Eveline D; Boven, Leonie A; Kondova, Ivanela; IJzerman, Ad P; Bajramovic, Jeffrey J

    2009-06-15

    Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

  12. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    PubMed

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  13. The CB1 receptor as an important mediator of hedonic reward processing.

    PubMed

    Friemel, Chris M; Zimmer, Andreas; Schneider, Miriam

    2014-09-01

    The endocannabinoid (ECB) system has emerged recently as a key mediator for reward processing. It is well known that cannabinoids affect appetitive learning processes and can induce reinforcing and rewarding effects. However, the involvement of the ECB system in hedonic aspects of reward-related behavior is not completely understood. With the present study, we investigated the modulatory role of the ECB system on hedonic perception, measured by the pleasure attenuated startle (PAS) paradigm for a palatable food reward. Here, a conditioned odor is thought to induce a pleasant affective state that attenuates an aversive reflex-the acoustic startle response. Modulatory effects of the CB1 receptor antagonist/inverse agonist SR1411716 and the cannabinoid agonist WIN 55 212-2 on PAS were examined in rats. PAS was also measured in CB1 receptor knockout (KO) and wild-type (WT) mice. Pharmacological inhibition as well as the absence of CB1 receptors was found to reduce PAS, whereas WIN 55 212-2 administration increased PAS. Finally, presentation of a conditioned reward cue was found to induce striatal FosB/ΔFosB expression in WT mice, but not in KO mice, indicating a reduced stimulation of reward-related brain regions in conditioned KO mice by odor presentation. We here show that in addition to our previous studies in rats, PAS may also serve as a valuable and suitable measure to assess hedonic processing in mice. Our data further indicate that the ECB system, and in particular CB1 receptor signaling, appears to be highly important for the mediation of hedonic aspects of reward processing.

  14. 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.

  15. 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

  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. The CB1 receptor as an important mediator of hedonic reward processing.

    PubMed

    Friemel, Chris M; Zimmer, Andreas; Schneider, Miriam

    2014-09-01

    The endocannabinoid (ECB) system has emerged recently as a key mediator for reward processing. It is well known that cannabinoids affect appetitive learning processes and can induce reinforcing and rewarding effects. However, the involvement of the ECB system in hedonic aspects of reward-related behavior is not completely understood. With the present study, we investigated the modulatory role of the ECB system on hedonic perception, measured by the pleasure attenuated startle (PAS) paradigm for a palatable food reward. Here, a conditioned odor is thought to induce a pleasant affective state that attenuates an aversive reflex-the acoustic startle response. Modulatory effects of the CB1 receptor antagonist/inverse agonist SR1411716 and the cannabinoid agonist WIN 55 212-2 on PAS were examined in rats. PAS was also measured in CB1 receptor knockout (KO) and wild-type (WT) mice. Pharmacological inhibition as well as the absence of CB1 receptors was found to reduce PAS, whereas WIN 55 212-2 administration increased PAS. Finally, presentation of a conditioned reward cue was found to induce striatal FosB/ΔFosB expression in WT mice, but not in KO mice, indicating a reduced stimulation of reward-related brain regions in conditioned KO mice by odor presentation. We here show that in addition to our previous studies in rats, PAS may also serve as a valuable and suitable measure to assess hedonic processing in mice. Our data further indicate that the ECB system, and in particular CB1 receptor signaling, appears to be highly important for the mediation of hedonic aspects of reward processing. PMID:24718372

  18. Mononuclear Phagocyte-Mediated Antifungal Immunity: The Role of Chemotactic Receptors and Ligands

    PubMed Central

    Swamydas, Muthulekha; Break, Timothy J.; Lionakis, Michail S.

    2015-01-01

    Over the past two decades, fungal infections have emerged as significant causes of morbidity and mortality in patients with hematological malignancies, hematopoietic stem cell or solid organ transplantation and acquired immunodeficiency syndrome. Besides neutrophils and CD4+ T lymphocytes, which have long been known to play an indispensable role in promoting protective antifungal immunity, mononuclear phagocytes are now being increasingly recognized as critical mediators of host defense against fungi. Thus, a recent surge of research studies has focused on understanding the mechanisms by which resident and recruited monocytes, macrophages and dendritic cells accumulate and become activated at the sites of fungal infection. Herein, we critically review how a variety of G-protein coupled chemoattractant receptors and their ligands mediate mononuclear phagocyte recruitment and effector function during infection by the most common human fungal pathogens. PMID:25715741

  19. Mononuclear phagocyte-mediated antifungal immunity: the role of chemotactic receptors and ligands.

    PubMed

    Swamydas, Muthulekha; Break, Timothy J; Lionakis, Michail S

    2015-06-01

    Over the past two decades, fungal infections have emerged as significant causes of morbidity and mortality in patients with hematological malignancies, hematopoietic stem cell or solid organ transplantation and acquired immunodeficiency syndrome. Besides neutrophils and CD4(+) T lymphocytes, which have long been known to play an indispensable role in promoting protective antifungal immunity, mononuclear phagocytes are now being increasingly recognized as critical mediators of host defense against fungi. Thus, a recent surge of research studies has focused on understanding the mechanisms by which resident and recruited monocytes, macrophages and dendritic cells accumulate and become activated at the sites of fungal infection. Herein, we critically review how a variety of G-protein coupled chemoattractant receptors and their ligands mediate mononuclear phagocyte recruitment and effector function during infection by the most common human fungal pathogens. PMID:25715741

  20. Alpha-adrenergic receptors mediate imipramine/alarm substance-induced reaction in rats.

    PubMed

    Abel, E L

    1994-08-01

    The mechanism of adverse imipramine-induced reactions (jitteriness, convulsions) was investigated by precipitating such reactions in rats with three injections (IP) of imipramine (5-40 mg/kg) at 24, 5, and 1 h before testing, and comparing their occurrence with comparable treatments using specific noradrenergic and serotonergic reuptake inhibitors [nortriptyline (10 or 30 mg/kg, IP), citalopram (0.5-5.0 mg/kg, IP)]. This initial study indicated that these reactions were mediated by imipramine's noradrenergic effects. Subsequent combinations of imipramine and an alpha 2 agonist (clonidine, 5 mg/kg) and antagonist (yohimbine, 2 mg/kg), and a beta-adrenergic antagonist (propranolol, 2 or 5 mg/kg) (all administered IP 0.5 h after the last injection of imipramine) suggested imipramine's adverse effects were mediated by alpha 2 receptors. The possible involvement of the locus ceruleus in these effects was considered.

  1. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation.

    PubMed

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-02-16

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved. PMID:26840091

  2. Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

    PubMed

    Anaparti, Vidyanand; Pascoe, Christopher D; Jha, Aruni; Mahood, Thomas H; Ilarraza, Ramses; Unruh, Helmut; Moqbel, Redwan; Halayko, Andrew J

    2016-08-01

    We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

  3. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation

    PubMed Central

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-01-01

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophagecell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved. PMID:26840091

  4. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation.

    PubMed

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-02-16

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved.

  5. Kappa opioid receptors on dopaminergic neurons are necessary for kappa-mediated place aversion.

    PubMed

    Chefer, Vladimir I; Bäckman, Cristina M; Gigante, Eduardo D; Shippenberg, Toni S

    2013-12-01

    Kappa-opioid receptor (KOR) agonists have dysphoric properties in humans and are aversive in rodents. This has been attributed to the activation of KORs within the mesolimbic dopamine (DA) system. However, the role of DA in KOR-mediated aversion and stress remains divisive as recent studies have suggested that activation of KORs on serotonergic neurons may be sufficient to mediate aversive behaviors. To address this question, we used conditional knock-out (KO) mice with KORs deleted on DA neurons (DAT(Cre/wt)/KOR(loxp/loxp), or DATCre-KOR KO). In agreement with previous findings, control mice (DAT(Cre/wt)/KOR(wt/wt) or WT) showed conditioned place aversion (CPA) to the systemically administered KOR agonist U69,593. In contrast, DATCre-KOR KO mice did not exhibit CPA with this same agonist. In addition, in vivo microdialysis showed that systemic U69,593 decreased overflow of DA in the nucleus accumbens (NAc) in WT mice, but had no effect in DATCre-KOR KO mice. Intra- ventral tegmental area (VTA) delivery of KORs using an adeno-associated viral gene construct, resulted in phenotypic rescue of the KOR-mediated NAc DA response and aversive behavior in DATCre-KOR KO animals. These results provide evidence that KORs on VTA DA neurons are necessary to mediate KOR-mediated aversive behavior. Therefore, our data, along with recent findings, suggest that the neuronal mechanisms of KOR-mediated aversive behavior may include both dopaminergic and serotonergic components. PMID:23921954

  6. <