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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 Receptors in Neurological and Cardiovascular Disorders

    PubMed Central

    Skaper, Stephen D.; Debetto, Patrizia; Giusti, Pietro

    2009-01-01

    P2X receptors are ATP-gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X7, have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in a channel pore permeable to molecules as large as 900 daltons. The P2X7 receptor was originally described in cells of hematopoietic origin, and mediates the influx of Ca2+ and Na+ and Ca2+ and Na+ ions as well as the release of proinflammatory cytokines. P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7 receptors provides an inflammatory stimulus, and P2X7 receptor-deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7 receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. Apoptotic cell death occurs in a number of vascular diseases, including atherosclerosis, restenosis, and hypertension, and may be linked to the release of ATP from endothelial cells, P2X7 receptor activation, proinflammatory cytokine production, and endothelial cell apoptosis. In this context, the P2X7 receptor may be viewed as a gateway of communication between the nervous, immune, and cardiovascular systems. PMID:20029634

  5. 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.

  6. 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.

  7. Extracellular ATP mediates necrotic cell swelling in SN4741 dopaminergic neurons through P2X7 receptors.

    PubMed

    Jun, Dong-Jae; Kim, Jaeyoon; Jung, Sang-Yong; Song, Ran; Noh, Ji-Hyun; Park, Yong-Soo; Ryu, Sung-Ho; Kim, Joung-Hun; Kong, Young-Yun; Chung, Jun-Mo; Kim, Kyong-Tai

    2007-12-28

    Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X(7) receptors (P2X(7)Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X(7)Rs antagonist, KN62, or by knock-down of P2X(7)Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X(7)Rs.

  8. Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

    PubMed Central

    Guo, Y; Mishra, A; Weng, T; Chintagari, N R; Wang, Y; Zhao, C; Huang, C; Liu, L

    2014-01-01

    Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar–capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/β-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/β-catenin signaling through stimulating glycogen synthase kinase-3β (GSK-3β) and proteasome. On the other hand, the activation of Wnt/β-catenin signaling by Wnt3a, GSK-3β inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/β-catenin signaling to prevent the AEC I death and restrict the severity of ALI. PMID:24922070

  9. The protective effect of resveratrol in the transmission of neuropathic pain mediated by the P2X7 receptor in the dorsal root ganglia.

    PubMed

    Xie, Jinyan; Liu, Shuangmei; Wu, Bing; Li, Guilin; Rao, Shenqiang; Zou, Lifang; Yi, Zhihua; Zhang, Chunping; Jia, Tianyu; Zhao, Shanhong; Schmalzing, Günther; Hausmann, Ralf; Nie, Hong; Li, Guodong; Liang, Shangdong

    2017-02-01

    The P2X7 receptor mediates afferent nerve activation and is related to chronic neuropathic pain. Resveratrol (RES) has also been reported to exhibit anti-inflammatory effects. In this study, we investigated the neuroprotective effect of RES on the transmission of neuropathic pain mediated by the P2X7 receptor. The P2X7 mRNA and protein expression levels in L4-L5 dorsal root ganglia (DRG)s of the chronic constriction injury (CCI) group were significantly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups. RES increased the threshold of thermal and mechanical hypersensitivity in rats with chronic neuropathic pain. The P2X7 mRNA and protein expression levels in the CCI + RES group were decreased compared with those in the CCI group. Our results showed that RES inhibited the upregulated co-expression of P2X7 and glial fibrillary acidic protein (GFAP, a marker of satellite glial cells) in satellite glial cells of DRG in the CCI group. The results demonstrated that the expression of GFAP was increased in the CCI group and that RES inhibited the upregulated expression of GFAP in the rats in the CCI group. In addition, the phosphorylation levels of p38 and extracellular regulated protein kinases (ERK)1/2 in the CCI group were markedly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups, whereas the phosphorylation levels of p38 and ERK1/2 in CCI + RES group were markedly lower than those observed in the CCI group. RES inhibited BzATP-activated currents in DRG non-neurons in the CCI rats. Our data provide evidence that RES may suppress the transmission of neuropathic pain mediated by the P2X7 receptor in the satellite glial cells of dorsal root ganglia.

  10. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases.

    PubMed

    Lopez-Castejon, Gloria; Theaker, Jill; Pelegrin, Pablo; Clifton, Andrew D; Braddock, Martin; Surprenant, Annmarie

    2010-08-15

    The ATP-gated P2X(7) receptor (P2X(7)R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X(7)R antagonists are considered to result from inhibition of P2X(7)R-induced release of proinflammatory cytokines from activated macrophages. However, P2X(7)Rs are also expressed in resting macrophages, suggesting that P2X(7)R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X(7)R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1beta and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X(7)R antagonists, absent from P2X(7)R(-/-) mouse macrophages, and not associated with cell death. Our results suggest P2X(7)R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.

  11. PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

    PubMed Central

    Tsao, H-K; Chiu, P-H; Sun, S H

    2013-01-01

    Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca2+ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca2+ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca2+ and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway. PMID:23907465

  12. The P2X7 Receptor-Interleukin-1 Liaison

    PubMed Central

    Giuliani, Anna Lisa; Sarti, Alba C.; Falzoni, Simonetta; Di Virgilio, Francesco

    2017-01-01

    Interleukin-1β (IL-1β) plays a central role in stimulation of innate immune system and inflammation and in several chronic inflammatory diseases. These include rare hereditary conditions, e.g., auto-inflammatory syndromes, as well as common pathologies, such as type II diabetes, gout and atherosclerosis. A better understanding of IL-1β synthesis and release is particularly relevant for the design of novel anti-inflammatory drugs. One of the molecules mainly involved in IL-1β maturation is the P2X7 receptor (P2X7R), an ATP-gated ion channel that chiefly acts through the recruitment of the NLRP3 inflammasome-caspase-1 complex. In this review, we will summarize evidence supporting the key role of the P2X7R in IL-1β production, with special emphasis on the mechanism of release, a process that is still a matter of controversy. Four different models have been proposed: (i) exocytosis via secretory lysosomes, (ii) microvesicles shedding from plasma membrane, (iii) release of exosomes, and (iv) passive efflux across a leaky plasma membrane during pyroptotic cell death. All these models involve the P2X7R. PMID:28360855

  13. P2X7 receptors stimulate AKT phosphorylation in astrocytes

    PubMed Central

    Jacques-Silva, Maria C; Rodnight, Richard; Lenz, Guido; Liao, Zhongji; Kong, Qiongman; Tran, Minh; Kang, Yuan; Gonzalez, Fernando A; Weisman, Gary A; Neary, Joseph T

    2004-01-01

    Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′-triphosphate (UTP) and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism. PMID:15023862

  14. Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

    PubMed

    Faria, R X; Cascabulho, C M; Reis, R A M; Alves, Luiz Anastácio

    2010-07-01

    The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

  15. P2X7 receptor knockout prevents streptozotocin-induced type 1 diabetes in mice.

    PubMed

    Vieira, Flávia Sarmento; Nanini, Hayandra Ferreira; Takiya, Christina Maeda; Coutinho-Silva, Robson

    2016-01-05

    Type 1 diabetes (T1D) is caused by autoimmune destruction of islet of Langerhans β-cells. P2X7 receptors (P2X7R) modulate proinflammatory immune responses by binding extracellular ATP, a classic 'danger signal'. Here, we evaluated whether the P2X7R has a role in T1D development. P2X7(-/-) mice are resistant to TD1 induction by streptozotocin (STZ) treatment, with no increase in blood glucose, decrease in insulin-positive cells, and pancreatic islet reduction, compared to WT (C57BL/6) mice. Also, the levels of proinflammatory mediators (IL-1β, IFN-γ and NO) did not increase after STZ treatment in P2X7(-/-) animals, with reduced infiltration of CD4(+), CD8(+), B220(+), CD11b(+) and CD11c(+) cells in the pancreatic lymph nodes. Treatment with a P2X7 antagonist mimicked the effect of P2X7 knockout, preventing STZ-induced diabetes. Our results show that the absence of the P2X7R provides resistance in the induction of diabetes in this model, and suggest that therapy targeting the P2X7R may be useful against clinical T1D.

  16. Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts

    PubMed Central

    Masin, Marianela; Young, Christopher; Lim, KoiNi; Barnes, Sara J; Xu, Xing Jian; Marschall, Viola; Brutkowski, Wojciech; Mooney, Elizabeth R; Gorecki, Dariusz C; Murrell-Lagnado, Ruth

    2012-01-01

    BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function. PMID:21838754

  17. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    PubMed

    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

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/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 Ca(2+)-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 Ca(2+) 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 Ca(2+) 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.

  18. 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

  19. Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

    PubMed Central

    Nadal-Nicolás, Francisco M.; Galindo-Romero, Caridad; Valiente-Soriano, Francisco J.; Barberà-Cremades, María; deTorre-Minguela, Carlos; Salinas-Navarro, Manuel; Pelegrín, Pablo; Agudo-Barriuso, Marta

    2016-01-01

    Axonal injury is a common feature of central nervous system insults that culminates with the death of the affected neurons, and an irreversible loss of function. Inflammation is an important component of the neurodegenerative process, where the microglia plays an important role by releasing proinflammatory factors as well as clearing the death neurons by phagocytosis. Here we have identified the purinergic signaling through the P2X7 receptor as an important component for the neuronal death in a model of optic nerve axotomy. We have found that in P2X7 receptor deficient mice there is a delayed loss of retinal ganglion cells and a decrease of phagocytic microglia at early times points after axotomy. In contralateral to the axotomy retinas, P2X7 receptor controlled the numbers of phagocytic microglia, suggesting that extracellular ATP could act as a danger signal activating the P2X7 receptor in mediating the loss of neurons in contralateral retinas. Finally, we show that intravitreal administration of the selective P2X7 receptor antagonist A438079 also delays axotomy-induced retinal ganglion cell death in retinas from wild type mice. Thus, our work demonstrates that P2X7 receptor signaling is involved in neuronal cell death after axonal injury, being P2X7 receptor antagonism a potential therapeutic strategy. PMID:27929040

  20. The effect of anions on the human P2X7 receptor.

    PubMed

    Kubick, Christoph; Schmalzing, Günther; Markwardt, Fritz

    2011-12-01

    P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.

  1. 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

  2. Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage.

    PubMed

    Cisneros-Mejorado, Abraham; Gottlieb, Miroslav; Cavaliere, Fabio; Magnus, Tim; Koch-Nolte, Friederich; Scemes, Eliana; Pérez-Samartín, Alberto; Matute, Carlos

    2015-05-01

    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.

  3. Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner

    PubMed Central

    Mishra, Amarjit; Chintagari, Narendranath Reddy; Guo, Yujie; Weng, Tingting; Su, Lijing; Liu, Lin

    2011-01-01

    Alveolar epithelium is composed of alveolar epithelial cells of type I (AEC I) and type II (AEC II). AEC II secrete lung surfactant by means of exocytosis. P2X7 receptor (P2X7R), a P2 purinergic receptor, has been implicated in the regulation of synaptic transmission and inflammation. Here, we report that P2X7R, which is expressed in AEC I but not AEC II, is a novel mediator for the paracrine regulation of surfactant secretion in AEC II. In primary co-cultures of AEC I and AEC II benzoyl ATP (BzATP; an agonist of P2X7R) increased surfactant secretion, which was blocked by the P2X7R antagonist Brilliant Blue G. This effect was observed in AEC II co-cultured with human embryonic kidney HEK-293 cells stably expressing rat P2X7R, but not when co-cultured with AEC I in which P2X7R was knocked down or in co-cultures of AEC I and AEC II isolated from P2X7R−/− mice. BzATP-mediated secretion involved P2Y2 receptor signaling because it was reduced by the addition of the ATP scavengers apyrase and adenosine deaminase and the P2Y2 receptor antagonist suramin. However, the stimulation with BzATP might also release other substances that potentially increase surfactant secretion as a greater stimulation of secretion was observed in AEC II incubated with BzATP when co-cultured with E10 or HEK-293-P2X7R cells than with ATP alone. P2X7R−/− mice failed to increase surfactant secretion in response to hyperventilation, pointing to the physiological relevance of P2X7R in maintaining surfactant homeostasis in the lung. These results suggest that the activation of P2X7R increases surfactant secretion by releasing ATP from AEC I and subsequently stimulating P2Y2 receptors in AEC II. PMID:21266468

  4. Radiosensitizing Effect of P2X7 Receptor Antagonist on Melanoma in vitro and in vivo.

    PubMed

    Tanamachi, Keisuke; Nishino, Keisuke; Mori, Natsuki; Suzuki, Toshihiro; Tanuma, Sei-Ichi; Abe, Ryo; Tsukimoto, Mitsutoshi

    2017-03-24

    Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

  5. 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

  6. 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

  7. ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury.

    PubMed

    Del Puerto, Ana; Fronzaroli-Molinieres, Laure; Perez-Alvarez, María José; Giraud, Pierre; Carlier, Edmond; Wandosell, Francisco; Debanne, Dominique; Garrido, Juan José

    2015-08-01

    Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

  8. Electrophysiological studies of upregulated P2X7 receptors in rat superior cervical ganglia after myocardial ischemic injury.

    PubMed

    Kong, Fanjun; Liu, Shuangmei; Xu, Changshui; Liu, Jun; Li, Guodong; Li, Guilin; Gao, Yun; Lin, Hong; Tu, Guihua; Peng, Haiying; Qiu, Shuyi; Fan, Bo; Zhu, Qicheng; Yu, Shicheng; Zheng, Chaoran; Liang, Shangdong

    2013-09-01

    Myocardial ischemic injury activates cardiac sympathetic afferent fibers and elicits a sympathoexcitatory reflex by exciting sympathetic efferent action, with resultant augmentation of myocardial oxygen consumption, leading to a vicious cycle of exaggerating myocardial ischemia. P2X7 receptor participates in the neuronal functions and the neurological disorders. This study examined the role of P2X7 receptor of superior cervical ganglia (SCG) in sympathoexcitatory reflex. Our results showed that the expression of P2X7 receptor at both mRNA and protein in SCG was increased after myocardial ischemic injury. P2X7 receptor agonists at the same concentration activated much larger amplitudes of the currents in the SCG neurons of myocardial ischemic rats than those in control rats. P2X7 receptor antagonist (brilliant blue G, BBG) significantly inhibited P2X7 receptor agonist-activated currents in the SCG neurons. Excessive phosphorylation of MAPK ERK1/2 upon the activation of P2X7 receptor might be a mechanism mediating the signal transduction after myocardial ischemic injury. Therefore, the sensitized P2X7 receptor in SCG was involved in the nociceptive transmission of sympathoexcitatory reflex induced by myocardial ischemic injury.

  9. Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery.

    PubMed

    Zheng, Bin; Lai, Renchun; Li, Jun; Zuo, Zhiyi

    2017-03-01

    Postoperative cognitive dysfunction worsens patient outcome after surgery. Neuroinflammation is a critical neuropathological process for it. We determined the role of P2X7 receptors, proteins that participate in inflammatory response, in the neuroinflammation induction after surgery, and whether the choice of volatile anesthetics affects its occurrence. Eight-week old C57BL/6J or P2X7 receptor knockout male mice were subjected to right carotid arterial exposure under anesthesia with 1.8% isoflurane, 2.5% sevoflurane or 10% desflurane. They were tested by Barnes maze and fear conditioning from 2weeks after the surgery. Hippocampus was harvested 6h, 24h and 7days after the surgery for immunohistochemical staining and Western blotting. Mice with surgery under anesthesia with isoflurane, sevoflurane or desflurane took longer than control mice to identify the target box 1 or 8days after the training sessions in Barnes maze. Mice anesthetized by isoflurane or sevoflurane, but not by desflurane, had less freezing behavior than control mice in fear conditioning test. Mice with surgery and anesthesia had increased ionized calcium binding adapter molecule 1 and interleukin 1β in the hippocampus but this increase was smaller in mice anesthetized with desflurane than mice anesthetized with isoflurane. Mice with surgery had increased P2X7 receptors and its downstream molecule caspase 1. Inhibition or knockout of P2X7 receptors attenuated surgery and anesthesia-induced neuroinflammation and cognitive impairment. We conclude that surgery under desflurane anesthesia may have reduced neuroinflammation and cognitive impairment compared with surgery under isoflurane anesthesia. P2X7 receptors may mediate the neuroinflammation and cognitive impairment after surgery.

  10. P2X7 receptors induce degranulation in human mast cells.

    PubMed

    Wareham, Kathryn J; Seward, Elizabeth P

    2016-06-01

    Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.

  11. 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

  12. P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway

    PubMed Central

    Gandelman, Mandi; Levy, Mark; Cassina, Patricia; Barbeito, Luis; Beckman, Joseph S

    2013-01-01

    The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis (ALS). Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 3′-O-(4-benzoyl)-ATP (BzATP). The P2X7 inhibitors BBG, oATP and KN-62 prevented BzATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons. PMID:23646980

  13. Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally infected mice.

    PubMed

    Darville, Toni; Welter-Stahl, Lynn; Cruz, Cristiane; Sater, Ali Abdul; Andrews, Charles W; Ojcius, David M

    2007-09-15

    Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1beta, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1beta secretion.

  14. 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

  15. 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.

  16. Human neutrophils do not express purinergic P2X7 receptors

    PubMed Central

    Martel-Gallegos, Guadalupe; Rosales-Saavedra, María T.; Reyes, Juan P.; Casas-Pruneda, Griselda; Toro-Castillo, Carmen; Pérez-Cornejo, Patricia

    2010-01-01

    It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X7 receptors (P2X7R) to elicit Ca2+ entry, production of reactive oxygen species (ROS), processing and release of pro-inflammatory cytokines, shedding of adhesion molecules and uptake of large molecules. However, the expression of P2X7R at the plasma membrane of neutrophils has also been questioned since these putative responses are not always reproduced. In this work, we used electrophysiological recordings to measure functional responses associated with the activation of membrane receptors, spectrofluorometric measurements of ROS production and ethidium bromide uptake to asses coupling of P2X7R activation to downstream effectors, immune-labelling of P2X7R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X7R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X7R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells—a model cell for human neutrophils. We concluded that P2X7R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered. PMID:21103213

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

    PubMed Central

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

    2016-01-01

    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

  18. Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

    PubMed

    Gangadharan, Vimal; Nohe, Anja; Caplan, Jeffrey; Czymmek, Kirk; Duncan, Randall L

    2015-01-01

    The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

  19. Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF-β1

    PubMed Central

    Oliveira, Suellen D'arc Santos; Nanini, Hayandra Ferreira; Savio, Luiz Eduardo Baggio; Waghabi, Mariana Caldas; Silva, Claudia Lucia Martins

    2014-01-01

    Schistosomiasis is a chronic inflammatory disease whose macrophages are involved in immunopathology modulation. Although P2X7 receptor signaling plays an important role in inflammatory responses mediated by macrophages, no reports have examined the role of P2X7 receptors in macrophage function during schistosomiasis. Thus, we evaluated P2X7 receptor function in peritoneal macrophages during schistosomiasis using an ATP-induced permeabilization assay and measurements of the intracellular Ca2+ concentration. ATP treatment induced significantly less permeabilization in macrophages from S. mansoni-infected mice than in control cells from uninfected animals. Furthermore, P2X7-mediated increases in intracellular Ca2+ levels were also reduced in macrophages from infected mice. TGF-β1 levels were increased in the peritoneal cavity of infected animals, and pretreatment of control macrophages with TGF-β1 reduced ATP-induced permeabilization, mimicking the effect of S. mansoni infection. Western blot and qRT-PCR data showed no difference in P2X7 protein and mRNA between uninfected, infected, and TGF-β1-treated groups. However, immunofluorescence analysis revealed reduced cell surface localization of P2X7 receptors in macrophages from infected and TGF-β1-treated mice compared to controls. Therefore, our data suggest that schistosomiasis reduces peritoneal macrophage P2X7 receptor signaling. This effect is likely due to the fact that infected mice have increased levels of TGF-β1, which reduces P2X7 receptor cell surface expression. PMID:25276050

  20. Ependymal cells along the lateral ventricle express functional P2X(7) receptors.

    PubMed

    Genzen, Jonathan R; Platel, Jean-Claude; Rubio, Maria E; Bordey, Angelique

    2009-09-01

    Ependymal cells line the cerebral ventricles and are located in an ideal position to detect central nervous system injury and inflammation. The signaling mechanisms of ependymal cells, however, are poorly understood. As extracellular adenosine 5'-triphosphate is elevated in the context of cellular damage, experiments were conducted to determine whether ependymal cells along the mouse subventricular zone (SVZ) express functional purinergic receptors. Using whole-cell patch clamp recording, widespread expression of P2X(7) receptors was detected on ependymal cells based on their antagonist sensitivity profile and absence of response in P2X(7) (-/-) mice. Immunocytochemistry confirmed the expression of P2X(7) receptors, and electron microscopy demonstrated that P2X(7) receptors are expressed on both cilia and microvilli. Ca(2+) imaging showed that P2X(7) receptors expressed on cilia are indeed functional. As ependymal cells are believed to function as partner cells in the SVZ neurogenic niche, P2X(7) receptors may play a role in neural progenitor response to injury and inflammation.

  1. Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, Plecoglossus altivelis.

    PubMed

    Li, Chang-Hong; Lu, Xin-Jiang; Li, Ming-Yun; Chen, Jiong

    2015-12-01

    Cathelicidins (CATHs) are a family of endogenous antimicrobial peptides that are capable of both direct bacteria-killing and immunomodulatory effects. P2X7 receptor (P2X7R) is a mediator of CATH in mammalian immune cells. Here, we studied the function and regulation of CATH in head kidney-derived monocytes/macrophages (MO/MФ) from ayu, Plecoglossus altivelis. We investigated the chemotaxis of MO/MФ in response to ayu CATH (PaCATH), and found that PaCATH had a dose-dependent effect on MO/MФ chemotaxis with the optimal concentration of 10.0 μg/ml. The qPCR and Western blot analysis revealed that PaCATH inhibited the expression of ayu P2X7R (PaP2X7R) at both mRNA and protein levels. Knockdown of the PaP2X7R expression in ayu MO/MФ by RNA interference not only significantly inhibited the chemotactic effect of PaCATH on MO/MФ, but also obviously reduced the effect of PaCATH on the phagocytosis, bacteria-killing, respiratory burst, and cytokine expression of ayu MO/MФ. Our study revealed that the immunomodulatory effect of fish CATH is mediated by P2X7R.

  2. The role of P2X7 receptors in a rodent PCP-induced schizophrenia model

    PubMed Central

    Koványi, Bence; Csölle, Cecilia; Calovi, Stefano; Hanuska, Adrienn; Kató, Erzsébet; Köles, László; Bhattacharya, Anindya; Haller, József; Sperlágh, Beáta

    2016-01-01

    P2X7 receptors (P2X7Rs) are ligand-gated ion channels sensitive to extracellular ATP. Here we examined for the first time the role of P2X7R in an animal model of schizophrenia. Using the PCP induced schizophrenia model we show that both genetic deletion and pharmacological inhibition of P2X7Rs alleviate schizophrenia-like behavioral alterations. In P2rx7+/+ mice, PCP induced hyperlocomotion, stereotype behavior, ataxia and social withdrawal. In P2X7 receptor deficient mice (P2rx7−/−), the social interactions were increased, whereas the PCP induced hyperlocomotion and stereotype behavior were alleviated. The selective P2X7 receptor antagonist JNJ-47965567 partly replicated the effect of gene deficiency on PCP-induced behavioral changes and counteracted PCP-induced social withdrawal. We also show that PCP treatment upregulates and increases the functional responsiveness of P2X7Rs in the prefrontal cortex of young adult animals. The amplitude of NMDA evoked currents recorded from layer V pyramidal neurons of cortical slices were slightly decreased by both genetic deletion of P2rx7 and by JNJ-47965567. PCP induced alterations in mRNA expression encoding schizophrenia-related genes, such as NR2A, NR2B, neuregulin 1, NR1 and GABA α1 subunit were absent in the PFC of young adult P2rx7−/− animals. Our findings point to P2X7R as a potential therapeutic target in schizophrenia. PMID:27824163

  3. Lipin-2 regulates NLRP3 inflammasome by affecting P2X7 receptor activation.

    PubMed

    Lordén, Gema; Sanjuán-García, Itziar; de Pablo, Nagore; Meana, Clara; Alvarez-Miguel, Inés; Pérez-García, M Teresa; Pelegrín, Pablo; Balsinde, Jesús; Balboa, María A

    2017-02-01

    Mutations in human LPIN2 produce a disease known as Majeed syndrome, the clinical manifestations of which are ameliorated by strategies that block IL-1β or its receptor. However the role of lipin-2 during IL-1β production remains elusive. We show here that lipin-2 controls excessive IL-1β formation in primary human and mouse macrophages by several mechanisms, including activation of the inflammasome NLRP3. Lipin-2 regulates MAPK activation, which mediates synthesis of pro-IL-1β during inflammasome priming. Lipin-2 also inhibits the activation and sensitization of the purinergic receptor P2X7 and K(+) efflux, apoptosis-associated speck-like protein with a CARD domain oligomerization, and caspase-1 processing, key events during inflammasome activation. Reduced levels of lipin-2 in macrophages lead to a decrease in cellular cholesterol levels. In fact, restoration of cholesterol concentrations in cells lacking lipin-2 decreases ion currents through the P2X7 receptor, and downstream events that drive IL-1β production. Furthermore, lipin-2-deficient mice exhibit increased sensitivity to high lipopolysaccharide doses. Collectively, our results unveil lipin-2 as a critical player in the negative regulation of NLRP3 inflammasome.

  4. Human P2X7 receptor activation induces the rapid shedding of CXCL16.

    PubMed

    Pupovac, Aleta; Foster, Christopher M; Sluyter, Ronald

    2013-03-22

    Activation of the purinergic P2X7 receptor by extracellular ATP induces the shedding of cell-surface molecules including the low-affinity IgE receptor, CD23 from leukocytes. CD23 is a known substrate of a disintegrin and metalloprotease (ADAM)10. The aim of the current study was to determine if P2X7 activation induced the shedding of the chemokine CXCL16, an ADAM10 substrate. Using immunolabelling and flow cytometry we demonstrate that human RPMI 8226 multiple myeloma B cells, which have been previously shown to express P2X7, also express CXCL16. Flow cytometric and ELISA measurements of ATP-induced loss of cell-surface CXCL16 showed that ATP treatment of RPMI 8226 cells induced the rapid shedding of CXCL16. Treatment of RPMI 8226 cells with the specific P2X7 antagonists, AZ10606120 and KN-62 impaired ATP-induced CXCL16 shedding by ~86% and ~90% respectively. RT-PCR demonstrated that ADAM10 is expressed in these cells and treatment of cells with the ADAM10 inhibitor, GI254023X, impaired ATP-induced CXCL16 shedding by ~87%. GI254023X also impaired P2X7-induced CD23 shedding by ∼57%. This data indicates that human P2X7 activation induces the rapid shedding of CXCL16 and that this process involves ADAM10.

  5. Mechanism of action of species-selective P2X7 receptor antagonists

    PubMed Central

    Michel, Anton D; Ng, Sin-Wei; Roman, Shilina; Clay, William C; Dean, David K; Walter, Daryl S

    2009-01-01

    Background and purpose: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X7 receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. Experimental approach: Antagonist effects at recombinant and chimeric P2X7 receptors were assessed by ethidium accumulation and radioligand-binding studies. Key results: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X7 receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X7 receptors and, for each compound, their potency estimates at human and dog P2X7 receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X7 receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X7 receptor. N2-(3,4-difluorophenyl)-N1-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373. Conclusions: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X7 receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X7 receptor at which antagonists can bind and affect receptor function. PMID:19309360

  6. 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

  7. 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

  8. P2X7 from j774 murine macrophages acts as a scavenger receptor for bacteria but not yeast.

    PubMed

    Pérez-Flores, Gabriela; Hernández-Silva, Cesar; Gutiérrez-Escobedo, Guadalupe; De Las Peñas, Alejandro; Castaño, Irene; Arreola, Jorge; Pérez-Cornejo, Patricia

    2016-12-02

    We studied the effects of extracellular ATP and Ca(2+) on uptake of bacteria (Staphylococcus aureus or Escherichia coli) and live yeast (Candida glabrata) by J774 macrophages to determine the role of endogenous P2X7 receptors in phagocytosis. Our findings show that phagocytosis of bio-particles coated with S. aureus or E. coli was blocked by ATP and the P2X7 receptor agonist BzATP, while yeast phagocytosis was not. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. To determine if P2X7-mediated Ca(2+) entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca(2+) with or without ATP. Ca(2+), in the absence of ATP, was required for engulfment of E. coli and C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus and E. coli regardless of Ca(2+), suggesting that Ca(2+) entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper-adherent C. glabrata mutants had an absolute requirement for extracellular Ca(2+) due to yeast adhesion to macrophages mediated by Ca(2+)-dependent adhesion proteins. We conclude that unstimulated P2X7 from J774 cells act as scavenger receptor for the uptake of S. aureus and E. coli but not of yeast; Ca(2+) entry via P2X7 receptors play no role in phagocytosis of S. aureus and E. coli; while the effect of Ca(2+) on C. glabrata phagocytosis was mediated by the adhesins Epa1, Epa6 and Epa7.

  9. The P2X7 Receptor Supports Both Life and Death in Fibrogenic Pancreatic Stellate Cells

    PubMed Central

    Haanes, Kristian A.; Schwab, Albrecht; Novak, Ivana

    2012-01-01

    The pancreatic stellate cells (PSCs) have complex roles in pancreas, including tissue repair and fibrosis. PSCs surround ATP releasing exocrine cells, but little is known about purinergic receptors and their function in PSCs. Our aim was to resolve whether PSCs express the multifunctional P2X7 receptor and elucidate how it regulates PSC viability. The number of PSCs isolated from wild type (WT) mice was 50% higher than those from the Pfizer P2X7 receptor knock out (KO) mice. The P2X7 receptor protein and mRNA of all known isoforms were expressed in WT PSCs, while KO PSCs only expressed truncated versions of the receptor. In culture, the proliferation rate of the KO PSCs was significantly lower. Inclusion of apyrase reduced the proliferation rate in both WT and KO PSCs, indicating importance of endogenous ATP. Exogenous ATP had a two-sided effect. Proliferation of both WT and KO cells was stimulated with ATP in a concentration-dependent manner with a maximum effect at 100 µM. At high ATP concentration (5 mM), WT PSCs, but not the KO PSCs died. The intracellular Ca2+ signals and proliferation rate induced by micromolar ATP concentrations were inhibited by the allosteric P2X7 receptor inhibitor az10606120. The P2X7 receptor-pore inhibitor A438079 partially prevented cell death induced by millimolar ATP concentrations. This study shows that ATP and P2X7 receptors are important regulators of PSC proliferation and death, and therefore might be potential targets for treatments of pancreatic fibrosis and cancer. PMID:23284663

  10. P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance.

    PubMed

    Tang, Xiao; Basavarajappa, Devaraj; Haeggström, Jesper Z; Wan, Min

    2015-08-01

    Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence-dependent endocytotic process. Both clathrin- and caveolae/lipid raft-mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

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

    PubMed

    Sluyter, Ronald; Vine, Kara L

    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.

  12. P2X7 receptor inhibition protects against ischemic acute kidney injury in mice.

    PubMed

    Yan, Yanli; Bai, Jianwen; Zhou, Xiaoxu; Tang, Jinhua; Jiang, Chunming; Tolbert, Evelyn; Bayliss, George; Gong, Rujun; Zhao, Ting C; Zhuang, Shougang

    2015-03-15

    Activation of the purinergic P2X7 receptor (P2X7R) has been associated with the development of experimental nephritis and diabetic and hypertensive nephropathy. However, its role in acute kidney injury (AKI) remains unknown. In this study, we examined the effects of P2X7R inhibition in a murine model of ischemia-reperfusion (I/R)-induced AKI using A438079, a selective inhibitor of P2X7R. At 24 h after I/R, mice developed renal dysfunction and renal tubular damage, which was accompanied by elevated expression of P2X7R. Early administration of A438079 immediately or 6 h after the onset of reperfusion protected against renal dysfunction and attenuated kidney damage whereas delayed administration of A438079 at 24 h after restoration of perfusion had no protective effects. The protective actions of A438079 were associated with inhibition of renal tubule injury and cell death and suppression of renal expression of monocyte chemotactic protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). Moreover, I/R injury led to an increase in phosphorylation (activation) of extracellular signal-regulated kinases 1/2 in the kidney; treatment with A438079 diminished this response. Collectively, these results indicate that early P2X7R inhibition is effective against renal tubule injury and proinflammatory response after I/R injury and suggest that targeting P2X7R may be a promising therapeutic strategy for treatment of AKI.

  13. P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

    PubMed Central

    Santana, Patrícia T.; Vieira, Flávia S.; da Graça, Carolyne Lalucha A. L.; Marques-da-Silva, Camila; Machado, Mariana N.; Caruso-Neves, Celso; Zin, Walter A.; Borojevic, Radovan; Coutinho-Silva, Robson

    2014-01-01

    Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes. PMID:25310682

  14. Neuronal Release of Cytokine IL-3 Triggered by Mechanosensitive Autostimulation of the P2X7 Receptor Is Neuroprotective

    PubMed Central

    Lim, Jason C.; Lu, Wennan; Beckel, Jonathan M.; Mitchell, Claire H.

    2016-01-01

    Mechanical strain due to increased pressure or swelling activates inflammatory responses in many neural systems. As cytokines and chemokine messengers lead to both pro-inflammatory and neuroprotective actions, understanding the signaling patterns triggered by mechanical stress may help improve overall outcomes. While cytokine signaling in neural systems is often associated with glial cells like astrocytes and microglia, the contribution of neurons themselves to the cytokine response is underappreciated and has bearing on any balanced response. Mechanical stretch of isolated neurons was previously shown to trigger ATP release through pannexin hemichannels and autostimulation of P2X7 receptors (P2X7Rs) on the neural membrane. Given that P2X7Rs are linked to cytokine activation in other cells, this study investigates the link between neuronal stretch and cytokine release through a P2X7-dependent pathway. Cytokine assays showed application of a 4% strain to isolated rat retinal ganglion cells (RGCs) released multiple cytokines. The P2X7R agonist BzATP also released multiple cytokines; Interleukin 3 (IL-3), TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 were released by both stimuli, with the release of IL-3 greatest with either stimuli. Stretch-dependent IL-3 release was confirmed with ELISA and blocked by P2X7R antagonists A438079 and Brilliant Blue G (BBG), implicating autostimulation of the P2X7R in stretch-dependent IL-3 release. Neuronal IL-3 release triggered by BzATP required extracellular calcium. The IL-3Rα receptor was expressed on RGCs but not astrocytes, and both IL-3Rα and IL-3 itself were predominantly expressed in the retinal ganglion cell layer of adult retinal sections, implying autostimulation of receptors by released IL-3. While the number of surviving ganglion cells decreased with time in culture, the addition of IL-3 protected against this loss of neurons. Expression of mRNA for IL-3 and IL-3Rα increased in rat

  15. 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.

  16. P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone.

    PubMed

    Messemer, Nanette; Kunert, Christin; Grohmann, Marcus; Sobottka, Helga; Nieber, Karen; Zimmermann, Herbert; Franke, Heike; Nörenberg, Wolfgang; Straub, Isabelle; Schaefer, Michael; Riedel, Thomas; Illes, Peter; Rubini, Patrizia

    2013-10-01

    Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)](i) transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

  17. Structural basis for subtype-specific inhibition of the P2X7 receptor

    SciTech Connect

    Karasawa, Akira; Kawate, Toshimitsu

    2016-12-09

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

  18. Structural basis for subtype-specific inhibition of the P2X7 receptor

    PubMed Central

    Karasawa, Akira; Kawate, Toshimitsu

    2016-01-01

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases. DOI: http://dx.doi.org/10.7554/eLife.22153.001 PMID:27935479

  19. Astrocyte-neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor-mediated release of glutamate and reactive oxygen species.

    PubMed

    Ficker, Christoph; Rozmer, Katalin; Kató, Erzsébet; Andó, Rómeó D; Schumann, Luisa; Krügel, Ute; Franke, Heike; Sperlágh, Beáta; Riedel, Thomas; Illes, Peter

    2014-10-01

    The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions.

  20. 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

  1. P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

    PubMed

    Figliuolo, Vanessa R; Savio, Luiz Eduardo Baggio; Safya, Hanaa; Nanini, Hayandra; Bernardazzi, Cláudio; Abalo, Alessandra; de Souza, Heitor S P; Kanellopoulos, Jean; Bobé, Pierre; Coutinho, Cláudia M L M; Coutinho-Silva, Robson

    2017-03-07

    P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB(low). Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

  2. On the role of P2X(7) receptors in dopamine nerve cell degeneration in a rat model of Parkinson's disease: studies with the P2X(7) receptor antagonist A-438079.

    PubMed

    Marcellino, Daniel; Suárez-Boomgaard, Diana; Sánchez-Reina, María Dolores; Aguirre, José A; Yoshitake, Takashi; Yoshitake, Shimako; Hagman, Beth; Kehr, Jan; Agnati, Luigi F; Fuxe, Kjell; Rivera, Alicia

    2010-06-01

    The role of the ATP-gated receptor, P2X(7), has been evaluated in the unilateral 6-OHDA rat model of Parkinson's disease using the P2X(7) competitive antagonist A-438079. Nigral P2X(7) immunoreactivity was mainly located in microglia but also in astroglia. A-438079 partially but significantly prevented the 6-OHDA-induced depletion of striatal DA stores. However, this was not associated with a reduction of DA cell loss. Blockade of P2X(7) receptors may represent a novel protective strategy for striatal DA terminals in Parkinson's disease and warrants further future investigation.

  3. Functional polymorphisms in the P2X7 receptor gene are associated with stress fracture injury.

    PubMed

    Varley, Ian; Greeves, Julie P; Sale, Craig; Friedman, Eitan; Moran, Daniel S; Yanovich, Ran; Wilson, Peter J; Gartland, Alison; Hughes, David C; Stellingwerff, Trent; Ranson, Craig; Fraser, William D; Gallagher, James A

    2016-03-01

    Military recruits and elite athletes are susceptible to stress fracture injuries. Genetic predisposition has been postulated to have a role in their development. The P2X7 receptor (P2X7R) gene, a key regulator of bone remodelling, is a genetic candidate that may contribute to stress fracture predisposition. The aim of this study is to evaluate the putative contribution of P2X7R to stress fracture injury in two separate cohorts, military personnel and elite athletes. In 210 Israeli Defense Forces (IDF) military conscripts, stress fracture injury was diagnosed (n = 43) based on symptoms and a positive bone scan. In a separate cohort of 518 elite athletes, self-reported medical imaging scan-certified stress fracture injuries were recorded (n = 125). Non-stress fracture controls were identified from these cohorts who had a normal bone scan or no history or symptoms of stress fracture injury. Study participants were genotyped for functional SNPs within the P2X7R gene using proprietary fluorescence-based competitive allele-specific PCR assay. Pearson's chi-squared (χ (2)) tests, corrected for multiple comparisons, were used to assess associations in genotype frequencies. The variant allele of P2X7R SNP rs3751143 (Glu496Ala-loss of function) was associated with stress fracture injury, whilst the variant allele of rs1718119 (Ala348Thr-gain of function) was associated with a reduced occurrence of stress fracture injury in military conscripts (P < 0.05). The association of the variant allele of rs3751143 with stress fractures was replicated in elite athletes (P < 0.05), whereas the variant allele of rs1718119 was also associated with reduced multiple stress fracture cases in elite athletes (P < 0.05). The association between independent P2X7R polymorphisms with stress fracture prevalence supports the role of a genetic predisposition in the development of stress fracture injury.

  4. Role of P2X7 and P2Y2 receptors on α-secretase-dependent APP processing: Control of amyloid plaques formation “in vivo” by P2X7 receptor

    PubMed Central

    Miras-Portugal, M. Teresa; Diaz-Hernandez, Juan I.; Gomez-Villafuertes, Rosa; Diaz-Hernandez, Miguel; Artalejo, Antonio R.; Gualix, Javier

    2015-01-01

    Amyloid precursor protein (APP) is expressed in a large variety of neural and non-neural cells. The balance between non-pathogenic and pathologic forms of APP processing, mediated by α-secretase and β-secretase respectively, remains a crucial step to understand β-amyloid, Aβ42 peptide, formation and aggregation that are at the origin of the senile plaques in the brain, a characteristic hallmark of Alzheimer's disease (AD). In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation. The in vivo approach was made possible by the use of J20 mice, a transgenic mouse model of familial Alzheimer's disease (FAD) expressing human APP mutant protein. This animal exhibits prominent amyloid plaques by six months of age. In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques. This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme. The in vivo findings corroborate the therapeutic potential of P2X7 antagonists in the treatment of FAD. PMID:25848496

  5. 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

  6. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.

    PubMed

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Hein, Martina; Petersen, Frank; Thon, Lutz; Adam, Dieter; Bulfone-Paus, Silvia

    2005-04-01

    Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

  7. Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

    PubMed Central

    Glaser, Talita; de Oliveira, Sophia La Banca; Cheffer, Arquimedes; Beco, Renata; Martins, Patrícia; Fornazari, Maynara; Lameu, Claudiana; Junior, Helio Miranda Costa; Coutinho-Silva, Robson; Ulrich, Henning

    2014-01-01

    Background Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo. Principal Findings P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis. Conclusions In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed. PMID:24798220

  8. 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

  9. Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.

    PubMed

    Corrêa, Gladys; Almeida Lindenberg, Carolina de; Moreira-Souza, Aline Cristina de Abreu; Savio, Luiz Eduardo Baggio; Takiya, Christina Maeda; Marques-da-Silva, Camila; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2017-04-01

    Activation of the purinergic P2X7 receptor by extracellular ATP (eATP) potentiates proinflammatory responses during infections by intracellular pathogens. Extracellular ATP triggers an antimicrobial response in macrophages infected with Toxoplasma gondii in vitro, suggesting that purinergic signaling may stimulate host defense mechanisms against toxoplasmosis. Here, we provide in vivo evidence in support of this hypothesis, by showing that P2X7(-/-) mice are more susceptible than P2X7(+/+) mice to acute infection by the RH strain of T. gondii, and that this phenomenon is associated with a deficient proinflammatory response. Four days post-infection, peritoneal washes from infected P2X7(-/-) mice had no or little increase in the levels of the proinflammatory cytokines IL-12, IL-1β, IFN-γ, and TNF-α, whose levels increased markedly in samples from infected P2X7(+/+) mice. Infected P2X7(-/-) mice displayed an increase in organ weight and histological alterations in some of the 'shock organs' in toxoplasmosis - the liver, spleen and mesenteric lymph nodes. The liver of infected P2X7(-/-) mice had smaller granulomas, but increased parasite load/granuloma. Our results confirm that the P2X7 receptor is involved in containing T. gondii spread in vivo, by stimulating inflammation.

  10. P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness.

    PubMed

    Jelassi, B; Chantôme, A; Alcaraz-Pérez, F; Baroja-Mazo, A; Cayuela, M L; Pelegrin, P; Surprenant, A; Roger, S

    2011-05-05

    ATP-gated P2X(7) receptors (P2X(7)R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X(7)R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X(7)R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X(7) receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca(2+)-activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X(7)R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X(7)R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X(7)R antagonists.

  11. Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells.

    PubMed

    Pupovac, Aleta; Geraghty, Nicholas J; Watson, Debbie; Sluyter, Ronald

    2015-01-01

    Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

  12. 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

  13. 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.

  14. R270C polymorphism leads to loss of function of the canine P2X7 receptor.

    PubMed

    Spildrejorde, Mari; Bartlett, Rachael; Stokes, Leanne; Jalilian, Iman; Peranec, Michelle; Sluyter, Vanessa; Curtis, Belinda L; Skarratt, Kristen K; Skora, Amanda; Bakhsh, Tahani; Seavers, Aine; McArthur, Jason D; Dowton, Mark; Sluyter, Ronald

    2014-07-15

    The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

  15. 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.

  16. Role of P2X7 Receptor in an Animal Model of Mania Induced by D-Amphetamine.

    PubMed

    Gubert, Carolina; Fries, Gabriel Rodrigo; Pfaffenseller, Bianca; Ferrari, Pâmela; Coutinho-Silva, Robson; Morrone, Fernanda Bueno; Kapczinski, Flávio; Battastini, Ana Maria Oliveira

    2016-01-01

    The objective of this study was to explore the association between the P2X7 purinergic receptor (P2X7R) and neuroinflammation using a preclinical model of acute bipolar mania. We analyzed the modulatory effects of P2X7R agonist (3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate, BzATP) and antagonists (brilliant blue, BBG and 3-[[5-(2,3 dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride, A438079) on assessments related to behavior (locomotor activity), neuroinflammation (interleukin-1 beta, IL-1β; tumor necrosis factor alpha, TNF-α; and interleukin- 6, IL-6), oxidative stress (thiobarbituric acid reactive substances, TBARS) and neuroplasticity (brain-derived neurotrophic factor, BDNF) markers in a pharmacological model of mania induced by acute and chronic treatment with D-amphetamine (AMPH) (2 mg/kg) in mice. An apparent lack of responsiveness to AMPH was observed in terms of the locomotor activity in animals with blocked P2X7R or with genetic deletion of P2X7R in knockout (P2X7R(-/-)) mice. Likewise, P2X7R participated in the AMPH-induced increase of the proinflammatory and excitotoxic environment, as demonstrated by the reversal of IL-1β, TNF-α, and TBARS levels caused by P2X7R blocking. Our results support the hypothesis that P2X7R plays a role in the neuroinflammation induced by AMPH in a preclinical model of mania, which could explain the altered behavior. The present data suggest that P2X7R may be a therapeutic target related to the neuroinflammation reported in bipolar disorder.

  17. Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

    PubMed Central

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-01-01

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

  18. 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

  19. 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

  20. P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid*

    PubMed Central

    Gu, Ben J.; Duce, James A.; Valova, Valentina A.; Wong, Bruce; Bush, Ashley I.; Petrou, Steven; Wiley, James S.

    2012-01-01

    Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1–5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1–10 mm tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells. PMID:22461619

  1. Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors.

    PubMed

    Michel, Anton D; Xing, Mengle; Thompson, Kyla M; Jones, Clare A; Humphrey, Patrick P A

    2006-03-18

    In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.

  2. Association of IFN-γ and P2X7 Receptor Gene Polymorphisms in Susceptibility to Tuberculosis Among Iranian Patients.

    PubMed

    Shamsi, Mahdi; Zolfaghari, Mohammad Reza; Farnia, Parissa

    2016-03-01

    Interferon-gamma (IFN-γ) and P2X7 receptor are crucial for host defence against mycobacterial infections. Recent studies have indicated that IFN-γ, IFN-γ receptor 1 (IFN-γR1) andP2X7 gene polymorphisms are associated with susceptibility to pulmonary tuberculosis (TB). However, the relationship between IFN-γ and P2X7 polymorphism and TB susceptibility remains inconclusive in Iranian population. For this reason, single nucleotide polymorphisms (SNPs) in IFN-γ (G+2109A), IFN-γR1 (G-611A) and P2X7 genes (at -762, 1513 position) in patients (n = 100) were assessed using PCR-RFLP. Data were analysed with SPSS version 18. For the 2109 loci of IFN-γ gene, the frequency of mutant alleles between patients and controls were not statistically significant. However, there was a significant difference between the TB patient and controls for -611 alleles of IFN-γR1 (P = 0.01). Additionally, the frequency of P2X7 gene polymorphisms (SNP-762 and 1513) between patients and controls was statistically significant. In conclusions, our study revealed a significant association of IFN-γR1 and P2X7 genes polymorphisms with risk of developing TB in Iranian population.

  3. 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.

  4. P2X7 receptor-pannexin 1 hemichannel association: effect of extracellular calcium on membrane permeabilization.

    PubMed

    Poornima, V; Madhupriya, M; Kootar, S; Sujatha, G; Kumar, Arvind; Bera, Amal Kanti

    2012-03-01

    Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2', 3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X(7)R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X(7)R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X(7)R-panx1, triggered by the removal of [Ca(2+)](o).

  5. Targeting P(2)X(7) receptor for the treatment of central post-stroke pain in a rodent model.

    PubMed

    Kuan, Yung-Hui; Shih, Hsi-Chien; Tang, Sung-Chun; Jeng, Jiann-Shing; Shyu, Bai-Chuang

    2015-06-01

    Stroke is a leading cause of death and disability in industrialized countries. Approximately 8-14% of stroke survivors suffer from central post-stroke pain (CPSP) when hemorrhagic stroke occurs in lateral thalamic regions, which severely affects their quality of life. Because the mechanisms of CPSP are not well understood, effective treatments have not been developed. In the present study, we tested the hypothesis that persistent CPSP is caused by P(2)X(7)receptor activation after brain tissue damage and subsequent elevations in inflammatory cytokines. A thalamic hemorrhagic rat model was used, characterized by thermal and mechanical allodynia that develops in the subacute to chronic phases upon CPSP onset. We found a significant increase in P(2)X(7) expression in reactive microglia/macrophages in thalamic peri-lesion tissues at 5 weeks post-hemorrhage. Thalamic P(2)X(7) receptors were directly involved in pain transmission and hypersensitivity. The systemic targeting of P(2)X(7) receptors during the acute stage of hemorrhage rescued abnormal pain behaviors and neuronal activity in the thalamocingulate pathway by reducing reactive microglia/macrophage aggregation and associated inflammatory cytokines. After CPSP onset, the targeting of interleukin-1β reversed abnormal pain sensitivity. The aberrant spontaneous thalamocortical oscillations in rats with CPSP were modulated by blocking P(2)X(7) receptors. Taken together, our results suggest that targeting P(2)X(7) may be bi-effective in the treatment of CPSP, as both a pain blocker and immunosuppressant that inhibits inflammatory damage to brain tissue. P(2)X(7)receptors may serve as a potential target to prevent the occurrence of CPSP and may be beneficial for the recovery of patients from stroke.

  6. 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

  7. 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

    2016-04-05

    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.

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

    PubMed

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

    2015-12-21

    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.

  9. 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

  10. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    PubMed

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  11. Recent Patents on Novel P2X7 Receptor Antagonists and Their Potential for Reducing Central Nervous System Inflammation

    PubMed Central

    Friedle, Scott A.; Curet, Marjorie A.; Watters, Jyoti J.

    2009-01-01

    Inflammation arises in the CNS from a number of neurodegenerative and oncogenic disorders, as well as from ischemic and traumatic brain injuries. These pathologies give rise to increased levels of extracellular adenine nucleotides which, via activation of a variety of cell surface P2 purinergic receptors, influence the inflammatory activities of responding immune cells. One P2 receptor subtype in particular, the P2X7 receptor, potentiates the release of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) from macrophage-like cells. It is also thought to contribute to secondary brain injury by inducing neuronal cell death. Therefore, antagonism of this receptor could have significant therapeutic impact on all disorders, not just CNS, to which excessive inflammatory activities contribute. The use of currently available P2X7 receptor antagonists for the treatment of CNS inflammation has been limited to the generally non-selective antagonists PPADS, oxidized ATP, Brilliant Blue G, suramin, calmidizolium, and KN-62. However, the recent patents and development of novel P2X7 receptor antagonists, as discussed in this review, will provide new tools both for clinical and research purposes. Here we discuss compounds for which patents have been applied since 2006, from the following categories: benzamide inhibitors, bicycloheteroaryl compounds, acylhdranzine antagonists, biaromatic P2X7 antagonists, heterocyclic compounds and amide derivatives, and aromatic amine antagonists. PMID:19705995

  12. 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

  13. Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.

    PubMed

    Sánchez-Nogueiro, Jesús; Marín-García, Patricia; Bustillo, Diego; Olivos-Oré, Luis Alcides; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2014-12-05

    The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 μM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 μM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.

  14. Characterization of (11)C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation.

    PubMed

    Territo, Paul R; Meyer, Jill A; Peters, Jonathan S; Riley, Amanda A; McCarthy, Brian P; Gao, Mingzhang; Wang, Min; Green, Mark A; Zheng, Qi-Huang; Hutchins, Gary D

    2017-03-01

    The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with (11)C. We report the initial physical and biologic characterization of this novel ligand. Methods:(11)C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity (11)C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min(-1)⋅nM(-1), 0.2547 ± 0.0155 min(-1), and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections

  15. Assessment of mercury chloride-induced toxicity and the relevance of P2X7 receptor activation in zebrafish larvae.

    PubMed

    Cruz, Fernanda Fernandes; Leite, Carlos Eduardo; Pereira, Talita Carneiro Brandão; Bogo, Maurício Reis; Bonan, Carla Denise; Battastini, Ana Maria Oliveira; Campos, Maria Martha; Morrone, Fernanda Bueno

    2013-09-01

    Zebrafish (Danio rerio) has been adopted as a model for behavioral, immunological and toxicological studies. Mercury is a toxic heavy metal released into the environment. There is evidence indicating that heavy metals can modulate ionotropic receptors, including the purinergic receptor P2X7. Therefore, this study evaluated the in vivo effects of acute exposure to mercury chloride (HgCl2) in zebrafish larvae and to investigate the involvement of P2X7R in mercury-related toxicity. Larvae survival was evaluated for 24 h after exposure to HgCl2, ATP or A740003. The combination of ATP (1 mM) and HgCl2 (20 μg/L) decreased survival when compared to ATP 1 mM. The antagonist A740003 (300 and 500 nM) increased the survival time, and reversed the mortality caused by ATP and HgCl2 in association. Quantitative real time PCR showed a decrease of P2X7R expression in the larvae treated with HgCl2 (20 μg/L). Evaluating the oxidative stress our results showed decreased CAT (catalase) activity and increased MDA (malondialdehyde) levels. Of note, the combination of ATP with HgCl2 showed an additive effect. This study provides novel evidence on the possible mechanisms underlying the toxicity induced by mercury, indicating that it is able to modulate P2X7R in zebrafish larvae.

  16. Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

    PubMed Central

    Seil, Michèle; Fontanils, Unai; Etxebarria, Irantzu Gorrono; Pochet, Stéphanie; Garcia-Marcos, Mikel; Marino, Aida

    2008-01-01

    ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X7 knockout mice or in cells pretreated with a P2X7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species. PMID:18581262

  17. 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.

  18. ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells.

    PubMed

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Koch-Nolte, Friedrich; Haag, Friedrich; Bulfone-Paus, Silvia

    2009-04-01

    Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

  19. Evaluation of P2X7 receptor expression in peripheral lymphocytes and immune profile from patients with indeterminate form of Chagas disease.

    PubMed

    Souza, Viviane do Carmo Gonçalves; Dos Santos, Joabel Tonellotto; Cabral, Fernanda Licker; Barbisan, Fernanda; Azevedo, Maria Isabel; Dias Carli, Luiz Felipe; de Avila Botton, Sonia; Dos Santos Jaques, Jeandre Augusto; Rosa Leal, Daniela Bitencourt

    2017-03-01

    Chagas disease (CD) is caused by Trypanosoma cruzi, an intracellular protozoan which is a potent stimulator of cell-mediated immunity. In the indeterminate form of CD (IFCD) a modulation between pro- and anti-inflammatory responses establishes a host-parasite adaptation. It was previously demonstrated that purinergic ecto-enzymes regulates extracellular ATP and adenosine levels, influencing immune and inflammatory processes during IFCD. In inflammatory sites ATP, as well as its degradation product, adenosine, function as signaling molecules and immunoregulators through the activation of purinergic receptors. In this work, it was analyzed the gene and protein expression of P2X7 purinergic receptor in peripheral lymphocytes and serum immunoregulatory cytokines from IFCD patients. Gene and protein expression of P2X7 receptor (P2X7R), and serum cytokines (IL-2, IL-10, IL-17 and IFN-γ) were unaltered. However, IFCD group showed significantly higher IL-4 and IL-6 levels while TNF-α was significantly decreased. These results indicate that imune profile of IFCD patients displays anti-inflammatory characteristics, consistent with the establishment of an immunomodulatory response. Further study about the molecular knowledge of P2X7R in IFCD is useful to clarify the participation of purinergic system in the regulatory mechanism which avoid the progression of CD.

  20. The ATP-Gated P2X7 Receptor As a Target for the Treatment of Drug-Resistant Epilepsy

    PubMed Central

    Beamer, Edward; Fischer, Wolfgang; Engel, Tobias

    2017-01-01

    Despite the progress made in the development of new antiepileptic drugs (AEDs), the biggest challenges that epilepsy presents to drug development have remained unchanged for the last 80 years: finding a treatment with potential for modifying disease progression and reducing the percentage of patients resistant to all pharmacological interventions. The mechanism of action of the majority of AEDs is based on blocking Na+ and/or Ca2+ channels, promotion of GABA or inhibition of glutamate signaling. In order for further progress to be made, however, a fuller picture of epilepsy will need to be considered, including changes to blood–brain barrier permeability, synaptic plasticity, network reorganization, and gliosis. In particular, brain inflammation has attracted much attention over recent years. Emerging evidence demonstrates a causal role for brain inflammation in lowering seizure thresholds and driving epileptogenesis. Consistent with this, intervening in pro-inflammatory cascades has shown promise in animal models of epilepsy, with clinical trials of anti-inflammatory agents already underway. The ATP-gated purinergic P2X7 receptor (P2X7) has been proposed as a novel drug target for a host of neurological conditions, including epilepsy. Constitutive expression of P2X7 in the CNS is mainly on microglia, but neuronal and astroglial expression has also been suggested. Its function as a gatekeeper of inflammation is most clearly understood, however, it also plays a number of other important roles pertinent to icto- and epileptogenesis: depolarization of the cell membrane, release of macromolecules, induction of apoptosis and synaptic reorganization. Changes in P2X7 expression have been reported following prolonged seizures (status epilepticus) and during chronic epilepsy in both experimental models and patients. While much of the early work focused on the study of P2X7 during status epilepticus, there is now mounting data showing involvement of this receptor during

  1. Contribution of the Purinergic Receptor P2X7 to Development of Lung Immunopathology during Influenza Virus Infection

    PubMed Central

    Ermler, Megan E.; Schotsaert, Michael; Gonzalez, Ma G.; Gillespie, Virginia; Lim, Jean K.; García-Sastre, Adolfo

    2017-01-01

    ABSTRACT An exacerbated immune response is one of the main causes of influenza-induced lung damage during infection. The molecular mechanisms regulating the fate of the initial immune response to infection, either as a protective response or as detrimental immunopathology, are not well understood. The purinergic receptor P2X7 is an ionotropic nucleotide-gated ion channel receptor expressed on immune cells that has been implicated in induction and maintenance of excessive inflammation. Here, we analyze the role of this receptor in a mouse model of influenza virus infection using a receptor knockout (KO) mouse strain. Our results demonstrate that the absence of the P2X7 receptor results in a better outcome to influenza virus infection characterized by reduced weight loss and increased survival upon experimental influenza challenge compared to wild-type mice. This effect was not virus strain specific. Overall lung pathology and apoptosis were reduced in virus-infected KO mice. Production of proinflammatory cytokines and chemokines such as interleukin-10 (IL-10), gamma interferon (IFN-γ), and CC chemokine ligand 2 (CCL2) was also reduced in the lungs of the infected KO mice. Infiltration of neutrophils and depletion of CD11b+ macrophages, characteristic of severe influenza virus infection in mice, were lower in the KO animals. Together, these results demonstrate that activation of the P2X7 receptor is involved in the exacerbated immune response observed during influenza virus infection. PMID:28351919

  2. Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7.

    PubMed

    Kaiser, M; Sobottka, H; Fischer, W; Schaefer, M; Nörenberg, W

    2014-09-01

    Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound library screen. In this study, a detailed characterization of the pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat, and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA, reduces Ca(2+) influx via human P2X7 (hP2X7) with an IC50 of 4.3 µM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO-PRO-1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiologic experiments revealed a noncompetitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1β. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacologic properties.

  3. Effect of P2X4 and P2X7 receptor antagonism on the pressure diuresis relationship in rats

    PubMed Central

    Menzies, Robert I.; Unwin, Robert J.; Dash, Ranjan K.; Beard, Daniel A.; Cowley Jr., Allen W.; Carlson, Brian E.; Mullins, John J.; Bailey, Matthew A.

    2013-01-01

    Reduced glomerular filtration, hypertension and renal microvascular injury are hallmarks of chronic kidney disease, which has a global prevalence of ~10%. We have shown previously that the Fischer (F344) rat has lower GFR than the Lewis rat, and is more susceptible to renal injury induced by hypertension. In the early stages this injury is limited to the pre-glomerular vasculature. We hypothesized that poor renal hemodynamic function and vulnerability to vascular injury are causally linked and genetically determined. In the present study, normotensive F344 rats had a blunted pressure diuresis relationship, compared with Lewis rats. A kidney microarray was then interrogated using the Endeavour enrichment tool to rank candidate genes for impaired blood pressure control. Two novel candidate genes, P2rx7 and P2rx4, were identified, having a 7− and 3− fold increased expression in F344 rats. Immunohistochemistry localized P2X4 and P2X7 receptor expression to the endothelium of the pre-glomerular vasculature. Expression of both receptors was also found in the renal tubule; however there was no difference in expression profile between strains. Brilliant Blue G (BBG), a relatively selective P2X7 antagonist suitable for use in vivo, was administered to both rat strains. In Lewis rats, BBG had no effect on blood pressure, but increased renal vascular resistance, consistent with inhibition of some basal vasodilatory tone. In F344 rats BBG caused a significant reduction in blood pressure and a decrease in renal vascular resistance, suggesting that P2X7 receptor activation may enhance vasoconstrictor tone in this rat strain. BBG also reduced the pressure diuresis threshold in F344 rats, but did not alter its slope. These preliminary findings suggest a physiological and potential pathophysiological role for P2X7 in controlling renal and/or systemic vascular function, which could in turn affect susceptibility to hypertension-related kidney damage. PMID:24187541

  4. 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.

  5. Resilience and reduced c-Fos expression in P2X7 receptor knockout mice exposed to repeated forced swim test.

    PubMed

    Boucher, A A; Arnold, J C; Hunt, G E; Spiro, A; Spencer, J; Brown, C; McGregor, I S; Bennett, M R; Kassiou, M

    2011-08-25

    There is considerable evidence suggesting genetic factors play an important role in the pathophysiology of depression, possibly by increasing susceptibility to repeated environmental stressors. Recent linkage studies have associated a polymorphism of the gene coding for the P2X7 receptor (P2X7R) with both major depressive disorder and bipolar disorder. Here we assessed whether P2X7 deletion affected the behavioural and neural response to repeated stress. P2X7R knockout (P2X7-/-) mice were subjected to the forced swim test for three consecutive days and neuronal activation in response to the third exposure was assessed using c-Fos immunohistochemistry. In addition, anxiety was evaluated in another group of P2X7-/- mice using the elevated plus maze (EPM) and light dark emergence (LDE) tests. Equivalent levels of immobility were observed in P2X7-/- mice and wild-type (WT) mice on the first exposure to forced swim, but much greater immobility was seen in WT mice on second and third exposures. This suggests that P2X7-/- mice exhibit an impaired adaptive coping response to repeated stress. Reinforcing this view, c-Fos expression in the dentate gyrus of the hippocampus and in the basolateral amygdala was seen in WT mice but not P2X7-/- mice following repeated forced swim. In addition, decreased locomotor activity was detected in P2X7-/- mice without any specific effects on anxiety in the LDE test. However, P2X7-/- mice showed greater anxiety-like behaviour in the EPM. These data suggest that the P2X7R may be involved in the adaptive mechanisms elicited by exposure to repeated environmental stressors that leads to the development of depression-like behaviours. This suggests that P2X7R antagonists may be useful therapeutics for the treatment of major depression, possibly by increasing resilience in the face of repeated stress.

  6. Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.

    PubMed

    Kimm-Brinson, K L; Moeller, P D; Barbier, M; Glasgow, H; Burkholder, J M; Ramsdell, J S

    2001-05-01

    We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin

  7. Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.

    PubMed Central

    Kimm-Brinson, K L; Moeller, P D; Barbier, M; Glasgow, H; Burkholder, J M; Ramsdell, J S

    2001-01-01

    We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin PMID:11401756

  8. 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.

  9. Lipopolysaccharide induces alveolar macrophage necrosis via CD14 and the P2x7 receptor leading to Interleukin-1α release

    PubMed Central

    Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Jones, Heather D.; Tumurkhuu, Gantsetseg; Zhang, Wenxuan; Wawrowsky, Kolja A.; Crother, Timothy R.; Arditi, Moshe

    2015-01-01

    SUMMARY Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca2+ influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration. PMID:25862090

  10. Lipopolysaccharide Induces Alveolar Macrophage Necrosis via CD14 and the P2X7 Receptor Leading to Interleukin-1α Release.

    PubMed

    Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Jones, Heather D; Tumurkhuu, Gantsetseg; Zhang, Wenxuan; Wawrowsky, Kolja A; Crother, Timothy R; Arditi, Moshe

    2015-04-21

    Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca(2+) influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

  11. Subfailure Overstretch Injury Leads to Reversible Functional Impairment and Purinergic P2X7 Receptor Activation in Intact Vascular Tissue

    PubMed Central

    Luo, Weifeng; Guth, Christy M.; Jolayemi, Olukemi; Duvall, Craig L.; Brophy, Colleen Marie; Cheung-Flynn, Joyce

    2016-01-01

    Vascular stretch injury is associated with blunt trauma, vascular surgical procedures, and harvest of human saphenous vein for use in vascular bypass grafting. A model of subfailure overstretch in rat abdominal aorta was developed to characterize surgical vascular stretch injury. Longitudinal stretch of rat aorta was characterized ex vivo. Stretch to the haptic endpoint, where the tissues would no longer lengthen, occurred at twice the resting length. The stress produced at this length was greater than physiologic mechanical forces but well below the level of mechanical disruption. Functional responses were determined in a muscle bath, and this subfailure overstretch injury led to impaired smooth muscle function that was partially reversed by treatment with purinergic receptor (P2X7R) antagonists. These data suggest that vasomotor dysfunction caused by subfailure overstretch injury may be due to the activation of P2X7R. These studies have implications for our understanding of mechanical stretch injury of blood vessels and offer novel therapeutic opportunities. PMID:27747211

  12. 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

    2016-03-16

    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.

  13. P2X7 Receptors as a Transducer in the Co-Occurrence of Neurological/Psychiatric and Cardiovascular Disorders: A Hypothesis

    PubMed Central

    Skaper, Stephen D.; Giusti, Pietro

    2009-01-01

    Background. Over-stimulation of the purinergic P2X7 receptor may bring about cellular dysfunction and injury in settings of neurodegeneration, chronic inflammation, as well as in psychiatric and cardiovascular diseases. Here we speculate how P2X7 receptor over-activation may lead to the co-occurrence of neurological and psychiatric disorders with cardiovascular disorders. Presentation. We hypothesize that proinflammatory cytokines, in particular interleukin-1β, are key players in the pathophysiology of neurological, psychiatric, and cardiovascular diseases. Critically, this premise is based on a role for the P2X7 receptor in triggering a rise in these cytokines. Given the broad distribution of P2X7 receptors in nervous, immune, and vascular tissue cells, this receptor is proposed as central in linking the nervous, immune, and cardiovascular systems. Testing. Investigate, retrospectively, whether a bidirectional link can be established between illnesses with a proinflammatory component (e.g., inflammatory and chronic neuropathic pain) and cardiovascular disease, for example, hypertension, and whether patients treated with anti-inflammatory drugs have a lower incidence of disease complications. Positive outcome would indicate a prospective study to evaluate therapeutic efficacy of P2X7 receptor antagonists. Implications. It should be stressed that sufficient direct evidence does not exist at present supporting our hypothesis. However, a positive outcome would encourage the further development of P2X7 receptor antagonists and their application to limit the co-occurrence of neurological, psychiatric, and cardiovascular disorders. PMID:20029625

  14. Lack of neuroprotection in the absence of P2X7 receptors in toxin-induced animal models of Parkinson's disease

    PubMed Central

    2011-01-01

    Background Previous studies indicate a role of P2X7 receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X7 receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD). Results mRNA encoding P2X7 and P2X4 receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X7 antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X7 receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X4 receptors was higher in P2X7 knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X7 knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. Conclusions We conclude that P2X7 receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD. PMID:21542899

  15. Evidence for associations between the purinergic receptor P2X7 (P2RX7) and toxoplasmosis

    PubMed Central

    Jamieson, Sarra E.; Peixoto-Rangel, Alba L.; Hargrave, Aubrey C.; de Roubaix, Lee-Anne; Mui, Ernest J.; Boulter, Nicola R.; Miller, E. Nancy; Fuller, Stephen J.; Wiley, James S.; Castellucci, Léa; Boyer, Kenneth; Peixe, Ricardo Guerra; Kirisits, Michael J.; de Souza Elias, Liliani; Coyne, Jessica J.; Correa-Oliveira, Rodrigo; Sautter, Mari; Smith, Nicholas C.; Lees, Michael P.; Swisher, Charles N.; Heydemann, Peter; Noble, A. Gwendolyn; Patel, Dushyant; Bardo, Dianna; Burrowes, Delilah; McLone, David; Roizen, Nancy; Withers, Shawn; Bahia-Oliveira, Lílian M. G.; McLeod, Rima; Blackwell, Jenefer M.

    2010-01-01

    Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus, and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong pro-inflammatory responses. Ligation of ATP by purinergic receptor P2X7, encoded by P2RX7, stimulates pro-inflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X7 plays a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z scores ±2.429; P= 0.015) between the derived C(+)G(−) allele (f= 0.68; OR= 2.06; 95% CI: 1.14–3.75) at SNP rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical sub-groups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0 to 4.25; 0.004

  16. miR-9 Mediates CALHM1-Activated ATP-P2X7R Signal in Painful Diabetic Neuropathy Rats.

    PubMed

    Liu, Wenjie; Ao, Qinying; Guo, Qulian; He, Wanyou; Peng, Liangyu; Jiang, Jun; Hu, Xiaoling

    2017-03-01

    In this study, we planned to illuminate the mechanisms of the expression and function of CALHM1 in painful diabetic neuropathy (PDN). PDN rat model was constructed. The expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons was detected. The correlation between the level of CALHM1 mRNA and 50 % PWT and the relationship between the expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons were statistically analyzed. The effect of miR-9 and CALHM1 on each other's expression in PDN rat spinal dorsal horn neurons were tested by qRT-PCR or Western blot. The co-culture system of neurons and glias from PDN rat spinal dorsal horn was constructed. The concentration of calcium and ATP as well as the expression of P2X7 receptor regulated by CALHM1 and miR-9 in PDN rat spinal dorsal horn neurons was measured. The results showed that the expression of CALHM1 was increased in PDN rat compared with controls, while its mRNA level was negatively correlated with 50 % PWT. miR-9, which was also upregulated in the spinal dorsal horn neurons of PDN rats, was positively correlated with the expression of CALHM1. The concentration of calcium and ATP as well as the expression of P2X7 receptor in glias was also increased in PDN rats. These increases could be reverted by inhibiting CALHM1 and/or miR-9. CALHM1 is involved in miR-9-mediated ATP-P2X7 pathway between neurons and glias in PDN rat.

  17. Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells.

    PubMed

    Wu, Pei-Yu; Lin, Yu-Chia; Chang, Chia-Ling; Lu, Hsing-Tsen; Chin, Chia-Hsuan; Hsu, Tsan-Ting; Chu, Dachen; Sun, Synthia H

    2009-06-01

    Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

  18. Silencing P2X7 receptor downregulates the expression of TCP-1 involved in lymphoma lymphatic metastasis.

    PubMed

    Jiang, Xudong; Mao, Wenjuan; Yang, Ziyi; Zeng, Jia; Zhang, Yi; Song, Yang; Kong, Ying; Ren, Shuangyi; Zuo, Yunfei

    2015-12-08

    P2X7R is an ATP-gated cation channel that participates in cell proliferation and apoptosis. TCP-1 assists with the protein folding. According to our previous research, the P2X7R has a potential role in P388D1 lymphoid neoplasm cells dissemination to peripheral lymph nodes. In order to make a further exploration about the probable mechanism, the lymph nodes which metastasized by P2X7R-silenced P388D1 cells or non-silenced cells were analyzed by 2DE and a MALDI-TOF-based proteomics approach. In the 64 proteins which were differentially expressed between two groups, TCP-1 was found to be significantly decreased in P2X7R shRNA group compared to controls. This correlation was also found in subsequent experiments in vivo and in vitro. The positive correlation between P2X7R and TCP-1 was also proved in both lymphoma and benign lymphadenopathy tissues from patients. It indicates that TCP-1 may be a crucial downstream molecular of P2X7R and plays a novel role in lymphoid neoplasm metastasis.

  19. Intra-Articular Blockade of P2X7 Receptor Reduces the Articular Hyperalgesia and Inflammation in the Knee Joint Synovitis Especially in Female Rats.

    PubMed

    Teixeira, Juliana Maia; Dias, Elayne Vieira; Parada, Carlos Amílcar; Tambeli, Cláudia Herrera

    2017-02-01

    Synovitis is a key factor in joint disease pathophysiology, which affects a greater proportion of women than men. P2X7 receptor activation contributes to arthritis, but whether it plays a role in articular inflammatory pain in a sex-dependent manner is unknown. We investigated whether the P2X7 receptor blockade in the knee joint of male and female rats reduces the articular hyperalgesia and inflammation induced by a carrageenan knee joint synovitis model. Articular hyperalgesia was quantified using the rat knee joint incapacitation test and the knee joint inflammation, characterized by the concentration of cytokines tumor necrosis factor-α, interleukin-1β, interleukin-6, and cytokine-induced neutrophil chemoattractant-1, and by neutrophil migration, was quantified using enzyme-linked immunosorbent assay and by myeloperoxidase enzyme activity measurement, respectively. P2X7 receptor blockade by the articular coadministration of selective P2X7 receptor antagonist A740003 with carrageenan significantly reduced articular hyperalgesia, pro-inflammatory cytokine concentrations, and myeloperoxidase activity induced by carrageenan injection into the knee joint of male and estrus female rats. However, a lower dose of P2X7 receptor antagonist was sufficient to significantly induce the antihyperalgesic and anti-inflammatory effects in estrus female but not in male rats. These results suggest that P2X7 receptor activation by endogenous adenosine 5'-triphosphate is essential to articular hyperalgesia and inflammation development in the knee joint of male and female rats. However, female rats are more responsive than male rats to the antihyperalgesic and anti-inflammatory effects induced by P2X7 receptor blockade.

  20. 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

  1. ATP induces NO production in hippocampal neurons by P2X(7) receptor activation independent of glutamate signaling.

    PubMed

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3')-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N(ω)-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

  2. ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

    PubMed Central

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C.; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity. PMID:23472093

  3. 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

  4. P2X7 receptors in body temperature, locomotor activity, and brain mRNA and lncRNA responses to sleep deprivation.

    PubMed

    Davis, Christopher J; Taishi, Ping; Honn, Kimberly A; Koberstein, John N; Krueger, James M

    2016-12-01

    The ionotropic purine type 2X7 receptor (P2X7R) is a nonspecific cation channel implicated in sleep regulation and brain cytokine release. Many endogenous rhythms covary with sleep, including locomotor activity and core body temperature. Furthermore, brain-hypothalamic cytokines and purines play a role in the regulation of these physiological parameters as well as sleep. We hypothesized that these parameters are also affected by the absence of the P2X7 receptor. Herein, we determine spontaneous expression of body temperature and locomotor activity in wild-type (WT) and P2X7R knockout (KO) mice and how they are affected by sleep deprivation (SD). We also compare hypothalamic, hippocampal, and cortical cytokine- and purine-related receptor and enzyme mRNA expressions before and after SD in WT and P2X7RKO mice. Next, in a hypothesis-generating survey of hypothalamic long noncoding (lnc) RNAs, we compare lncRNA expression levels between strains and after SD. During baseline conditions, P2X7RKO mice had attenuated temperature rhythms compared with WT mice, although locomotor activity patterns were similar in both strains. After 6 h of SD, body temperature and locomotion were enhanced to a greater extent in P2X7RKO mice than in WT mice during the initial 2-3 h after SD. Baseline mRNA levels of cortical TNF-α and P2X4R were higher in the KO mice than WT mice. In response to SD, the KO mice failed to increase hypothalamic adenosine deaminase and P2X4R mRNAs. Further, hypothalamic lncRNA expressions varied by strain, and with SD. Current data are consistent with a role for the P2X7R in thermoregulation and lncRNA involvement in purinergic signaling.

  5. P2X7 receptor antagonists protect against N-methyl-D-aspartic acid-induced neuronal injury in the rat retina.

    PubMed

    Sakamoto, Kenji; Endo, Kanako; Suzuki, Taishi; Fujimura, Kyosuke; Kurauchi, Yuki; Mori, Asami; Nakahara, Tsutomu; Ishii, Kunio

    2015-06-05

    Activation of N-methyl-d-aspartic acid (NMDA) receptors followed by a large Ca(2+) influx is thought to be a mechanism of glaucoma-induced neuronal cell death. It is possible that damage-associated molecular patterns leak from injured cells, such as adenosine triphosphate, causing retinal ganglion cell death in glaucoma. In the present study, we histologically investigated whether antagonists of the P2X7 receptor protected against NMDA-induced retinal injury in the rat in vivo. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal injection of NMDA. We used A438079 (3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine) and brilliant blue G as P2X7 receptor antagonists. Upon morphometric evaluation 7 days after an intravitreal injection (200 nmol/eye), NMDA-induced cell loss was apparent in the ganglion cell layer. Intravitreal A438079 (50 pmol/eye) simultaneously injected with NMDA and intraperitoneal brilliant blue G (50 mg/kg) administered just before the NMDA injection as well as 24 and 48h after significantly reduced cell loss. In addition, A438079 decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells 12h after NMDA injection. P2X7 receptors were immunolocalized in the ganglion cell layer and the inner and outer plexiform layers, whereas the immunopositive P2X7 receptor signal was not detected on the Iba1-positive microglial cells that infiltrated the retina 12h after NMDA injection. The present study shows that stimulation of the P2X7 receptor is involved in NMDA-induced histological damage in the rat retina in vivo. P2X7 receptor antagonists may be effective in preventing retinal diseases caused by glutamate excitotoxicity, such as glaucoma and retinal artery occlusion.

  6. Lack of the P2X7 receptor protects against AMD-like defects and microparticle accumulation in a chronic oxidative stress-induced mouse model of AMD.

    PubMed

    Carver, Kyle A; Lin, C M; Bowes Rickman, Catherine; Yang, Dongli

    2017-01-01

    The P2X7 receptor (P2X7R) is an ATP-gated ion channel that is a key player in oxidative stress under pathological conditions. The P2X7R is expressed in the retinal pigmented epithelium (RPE) and neural retina. Chronic oxidative stress contributes to the pathogenesis of age-related macular degeneration (AMD). Mice lacking Cu, Zn superoxide dismutase (Sod1) developed chronic oxidative stress as well as AMD-like features, but whether the P2X7R plays a causative role in oxidative stress-induced AMD is unknown. Thus, the main purpose of this study was to test if concurrent knockout (KO) of P2X7R could block AMD-like defects seen in Sod1 KO mice. Using multiple approaches, we demonstrate that Sod1 KO causes AMD-like defects, including positive staining for oxidative stress markers, 3-nitrotyrosine and carboxymethyl lysine, thinning of the RPE and retina, thickening of Bruch's membrane, presence of basal laminar and linear deposits, RPE barrier disruption and accumulation of microglia/macrophages. Moreover, we find that Sod1 KO mice accumulate more microparticles (MPs) within RPE/choroid tissues. Concurrent KO of the P2X7R protects against AMD-like defects and MP accumulation in Sod1 KO mice. Together, we show for the first time, that deficiency of P2X7R prevents in vivo oxidative stress-induced accumulation of MPs and AMD-like defects. This work could potentially lead to novel therapies for AMD and other oxidative stress-driven diseases.

  7. Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis.

    PubMed

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

    2014-07-01

    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.

  8. 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.

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

    PubMed

    Zhang, Yuanyuan; Yuan, Fahuan; Cao, Xuejiao; Zhai, Zhifang; GangHuang; 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.

  10. P2X7 receptor as a novel drug delivery system to increase the entrance of hydrophilic drugs into cells during photodynamic therapy.

    PubMed

    Pacheco, Paulo Anastácio Furtado; Ferreira, Leonardo Braga Gomes; Mendonça, Leonardo; Ferreira, Dinarte Neto M; Salles, Juliana Pimenta; Faria, Robson Xavier; Teixeira, Pedro Celso Nogueira; Alves, Luiz Anastacio

    2016-08-01

    The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319 Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900 Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5 μM MB for 15 min promoted the rate of entry of MB into the cytoplasm to 4.7 %. However, treatment with 5 μM MB and 1 mM ATP for the same amount of time increased this rate to 90.2 %. However, this effect was inhibited by pretreatment with a P2X7 antagonist. We used peritoneal macrophages and a cell line that does not express P2X7R as controls. These cells were more resistant to PDT with MB under the same experimental conditions. Taken together, these results suggest the use of the pore associated with P2X7R as a drug delivery system to increase the passage of hydrophilic drugs into cells that express this receptor, thus facilitating PDT.

  11. 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.

  12. The effect of P2X7 receptor activation on nuclear factor-κB phosphorylation induced by status epilepticus in the rat hippocampus.

    PubMed

    Kim, Ji-Eun; Kim, Duk-Soo; Jin Ryu, Hea; Il Kim, Won; Kim, Min-Ju; Won Kim, Dae; Young Choi, Soo; Kang, Tea-Cheon

    2013-06-01

    Nuclear factor-kappa B (NFκB) signal is essential for neuronal survival and its activation may protect neuron against various stimuli. Since purinergic signals activate NFκB through the P2X7 receptor, we investigated the distinct pattern of NF-κB phosphorylation in neurons by P2X7 receptor activation following status epilepticus (SE) in an effort to understand the role of P2X7 receptor in epileptogenic insult. In non-SE animals, 2'(3')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP, a P2X7R agonist) treatment increased only p52-Ser869 NF-κB phosphorylation in neuron. Following SE, p52-Ser865, p52-Ser869, p65-Ser276, p65-Ser311, p65-Ser468, and p65-Ser529 NF-κB phosphorylation was significantly decreased in CA1 and CA3 neurons. However, BzATP treatment prevented reductions in p65-Ser276, p65-Ser311, p65-Ser529, and p52-Ser869 NF-κB phosphorylations in CA1 and/or CA3 neurons induced by SE. Furthermore, BzATP treatment reduced SE-induced p65-Ser311, p65-Ser468, p65-Ser536, and p52-Ser869 NF-κB phosphorylations in astrocytes. These findings indicate that P2X7 functions may be involved in the regulation of SE-induced reactive astrocytes and neuronal degeneration via NF-κB phosphorylations in response to pilocarpine-induced SE in the rat hippocampus.

  13. Adipose-Derived Stem Cells Suppress Inflammation Induced by IL-1β through Down-Regulation of P2X7R Mediated by miR-373 in Chondrocytes of Osteoarthritis

    PubMed Central

    Jin, Rilong; Shen, Miaoda; Yu, Liedao; Wang, Xuanwei; Lin, Xiangjin

    2017-01-01

    Adipose-derived stem cells (ADSCs) were previously considered to have an anti-inflammatory effect, and Interleukin-1β (IL-1β) was found to be a pro-inflammatory factor in chondrocytes, but the mechanism underlying ADSCs and IL-1β is unclear. In this study, we investigate whether P2X7 receptor (P2X7R) signalling, regulated by microRNA 373 (miR-373), was involved in the ADSCs and IL-1β mediated inflammation in osteoarthritis (OA). Chondrocytes were collected from 20 OA patients and 20 control participants, and ADSCs were collected from patients who had undergone abdominal surgery. The typical surface molecules of ASDCs were detected by flow cytometry. The level of nitric oxide (NO) was determined by Griess reagent. Concentrations of prostaglandin E2 (PGE2), interleukin 6 (IL-6), matrix metallopeptidase 3 (MMP-3) were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of IL-6, MMP-3, miR-373 and P2X7R were determined by real-time polymerase chain reaction (PCR), and Western blot was used to detect the protein expression of P2X7R. The typical potential characters of ADSCs were verified. In chondrocytes or OA tissues, the miR-373 expression level was decreased, but the P2X7R expression was increased. IL-1β stimulation increased the level of inflammatory factors in OA chondrocytes, and ADSCs co-cultured with IL-1β-stimulated chondrocytes decreased the inflammation. OA chondrocytes transfected with the miR-373 inhibitor increased the inflammation level. The miR-373 mimic suppressed the inflammation by targeting P2X7R and regulated its expression, while its effect was reversed by overexpression of P2X7R. IL-1β induced inflammation in OA chondrocytes, while ADSCs seemed to inhibit the expression of P2X7R that was regulated by miR-373 and involved in the anti-inflammatory process in OA. PMID:28343378

  14. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  15. P2X7 Receptor Inhibition Increases CNTF in the Subventricular Zone, But Not Neurogenesis or Neuroprotection After Stroke in Adult Mice

    PubMed Central

    Kang, Seong Su; Keasey, Matthew Phillip

    2013-01-01

    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. PMID:24312160

  16. 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.

  17. 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.

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

    PubMed

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

    2001-10-01

    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.

  19. 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

  20. 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

  1. 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.

  2. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  3. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach

    PubMed Central

    Massicot, France; Hache, Guillaume; David, Ludivine; Chen, Dominique; Leuxe, Charlotte; Garnier-Legrand, Laure; Rat, Patrice; Laprévote, Olivier; Coudoré, François

    2013-01-01

    Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2–200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain. PMID:23826152

  4. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

    PubMed

    Massicot, France; Hache, Guillaume; David, Ludivine; Chen, Dominique; Leuxe, Charlotte; Garnier-Legrand, Laure; Rat, Patrice; Laprévote, Olivier; Coudoré, François

    2013-01-01

    Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain.

  5. Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.

    PubMed

    Yang, Yan; Li, Hui; Li, Ting-Ting; Luo, Hao; Gu, Xi-Yao; Lü, Ning; Ji, Ru-Rong; Zhang, Yu-Qiu

    2015-05-20

    Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.

  6. 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.

  7. P2X7 Receptor Expression in Peripheral Blood Monocytes Is Correlated With Plasma C-Reactive Protein and Cytokine Levels in Patients With Type 2 Diabetes Mellitus: a Preliminary Report.

    PubMed

    Wu, Hong; Nie, Yijun; Xiong, Huangui; Liu, Shuangmei; Li, Guilin; Huang, An; Guo, Lili; Wang, Shouyu; Xue, Yun; Wu, Bing; Peng, Lichao; Song, Miaomiao; Li, Guodong; Liang, Shangdong

    2015-12-01

    Chronic inflammation plays a major role in development of type 2 diabetes mellitus (T2DM). C-reactive protein (CRP) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) are directly involved in the occurrence of insulin resistance. Increased extracellular ATP levels can amplify the inflammatory response in vivo via the P2X7 receptor. The present study aimed to assess the relationship between P2X7 receptor expression in human peripheral blood monocytes and plasma levels of TNF-α, IL-1β, and CRP in T2DM patients. The results showed the association of increased P2X7 receptor expression of monocytes with high serum CRP, TNF-α, and IL-1β levels. TNF-α and IL-1β levels were lowest in healthy subjects; in T2DM patients, these inflammatory markers were less abundant in individuals with normal CRP levels compared to those with high CRP contents. In contrast, IL-10 levels in T2DM patients with high CRP levels were dramatically decreased. P2X7 receptor expression in monocytes from T2DM patients with high CRP levels was significantly increased in comparison with healthy individuals and T2DM patients with normal CRP levels. These findings indicated that P2X7 receptor in peripheral blood monocytes may be involved in the pathological changes of T2DM, particularly affecting patients with high CRP levels.

  8. 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

  9. Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

    PubMed Central

    Minns, Martin S.; Teicher, Gregory; Rich, Celeste B.; Trinkaus-Randall, Vickery

    2017-01-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein–labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. PMID:26683661

  10. The Selective Antagonism of P2X7 and P2Y1 Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

    PubMed Central

    Maraula, Giovanna; Lana, Daniele; Coppi, Elisabetta; Gentile, Francesca; Mello, Tommaso; Melani, Alessia; Galli, Andrea; Giovannini, Maria Grazia; Pedata, Felicita; Pugliese, Anna Maria

    2014-01-01

    Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG. PMID:25526634

  11. P2X7 deficiency attenuates hypertension and renal injury in deoxycorticosterone acetate-salt hypertension.

    PubMed

    Ji, Xu; Naito, Yukiko; Weng, Huachun; Endo, Kosuke; Ma, Xiao; Iwai, Naoharu

    2012-10-15

    The P2X(7) receptor is a ligand-gated ion channel, and genetic variations in the P2X(7) gene significantly affect blood pressure. P2X(7) receptor expression is associated with renal injury and inflammatory diseases. Uninephrectomized wild-type (WT) and P2X(7)-deficient (P2X(7) KO) mice were subcutaneously implanted with deoxycorticosterone acetate (DOCA) pellets and fed an 8% salt diet for 18 days. Their blood pressure was assessed by a telemetry system. The mice were placed in metabolic cages, and urine was collected for 24 h to assess renal function. After 18 days of DOCA-salt treatment, P2X(7) mRNA and protein expression increased in WT mice. Blood pressure in P2X(7) KO mice was less than that of WT mice (mean systolic blood pressure 133 ± 3 vs. 150 ± 2 mmHg). On day 18, urinary albumin excretion was lower in P2X(7) KO mice than in WT mice (0.11 ± 0.07 vs. 0.28 ± 0.07 mg/day). Creatinine clearance was higher in P2X(7) KO mice than in WT mice (551.53 ± 65.23 vs. 390.85 ± 32.81 μl·min(-1)·g renal weight(-1)). Moreover, renal interstitial fibrosis and infiltration of immune cells (macrophages, T cells, B cells, and leukocytes) were markedly attenuated in P2X(7) KO mice compared with WT mice. The levels of IL-1β, released by macrophages, in P2X(7) KO mice had decreased dramatically compared with that in WT mice. These results strongly suggest that the P2X(7) receptor plays a key role in the development of hypertension and renal disease via increased inflammation, indicating its potential as a novel therapeutic target.

  12. 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

  13. 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.

  14. P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment.

    PubMed

    Young, Christopher N J; Brutkowski, Wojciech; Lien, Chun-Fu; Arkle, Stephen; Lochmüller, Hanns; Zabłocki, Krzysztof; Górecki, Dariusz C

    2012-05-01

    Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.

  15. P2X7-pannexin-1 and amyloid β-induced oxysterol input in human retinal cell: Role in age-related macular degeneration?

    PubMed

    Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Leguillier, Teddy; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

    2016-08-01

    Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid β involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid β/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid β incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid β induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.

  16. Porphyromonas gingivalis fimbriae dampen P2X7-dependent IL-1β secretion

    PubMed Central

    Morandini, Ana Carolina; Ramos-Junior, Erivan S.; Potempa, Jan; Nguyen, Ky-Anh; Oliveira, Ana Carolina; Bellio, Maria; Ojcius, David M.; Scharfstein, Julio; Coutinho-Silva, Robson

    2014-01-01

    Porphyromonas gingivalis is a major contributor to the pathogenesis of periodontitis, an infection-driven inflammatory disease that leads to bone destruction. This pathogen stimulates pro-IL-1β synthesis but not mature IL-1β secretion, unless the P2X7 receptor is activated by extracellular ATP. Here, we investigated the role of Pg fimbriae in eATP-induced IL-1β release. Bone marrow derived macrophages (BMDMs) from wild type (WT) or P2X7-deficient mice were infected with Pg (strain 381) or isogenic fimbriae deficient (strain DPG3) with or without subsequent eATP stimulation. DPG3 induced higher IL-1β secretion after eATP stimulation compared to 381 in WT BMDMs, but not in P2X7-deficient cells. This mechanism was dependent of K+ efflux and Ca2+-iPLA2 activity. Accordingly, non-fimbriated Pg failed to inhibit apoptosis via eATP/P2X7-pathway. Furthermore, Pg-driven stimulation of IL-1β was TLR2- and MyD88-dependent, and irrespective of fimbriae expression. Fimbriae-dependent down-modulation of IL-1β was selective, as levels of other cytokines remained unaffected by P2X7 deficiency. Confocal microscopy demonstrated the presence of discrete P2X7 expression in the absence of Pg stimulation which was enhanced by 381-stimulated cells. Notably, DPG3-infected macrophages revealed a distinct pattern of P2X7 receptor expression with a markedly foci formation. Collectively, these data demonstrate that eATP-induced IL-1β secretion is impaired by Pg fimbriae in a P2X7-dependent manner. PMID:24925032

  17. Inhibition of chlamydial infectious activity due to P2X7R-dependent phospholipase D activation.

    PubMed

    Coutinho-Silva, Robson; Stahl, Lynn; Raymond, Marie-Noëlle; Jungas, Thomas; Verbeke, Philippe; Burnstock, Geoffrey; Darville, Toni; Ojcius, David M

    2003-09-01

    Chlamydia trachomatis survives within host cells by inhibiting fusion between Chlamydia vacuoles and lysosomes. We show here that treatment of infected macrophages with ATP leads to killing of chlamydiae through ligation of the purinergic receptor, P2X(7)R. Chlamydial killing required phospholipase D (PLD) activation, as PLD inhibition led to rescue of chlamydiae in ATP-treated macrophages. However, there was no PLD activation nor chlamydial killing in ATP-treated P2X(7)R-deficient macrophages. P2X(7)R ligation exerts its effects by promoting fusion between Chlamydia vacuoles and lysosomes. P2X(7)R stimulation also resulted in macrophage death, but fusion with lysosomes preceded macrophage death and PLD inhibition did not prevent macrophage death. These results suggest that P2X(7)R ligation leads to PLD activation, which is directly responsible for inhibition of infection.

  18. Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor–Dependent Death of Retinal Endothelial Cells

    PubMed Central

    Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Dubyak, George R.; Kern, Timothy S.; Matsuyama, Shigemi; Subauste, Carlos S.

    2016-01-01

    Purpose Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox. Methods Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry. Results Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner. Conclusions Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration. PMID:27893093

  19. 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

  20. Expression of the apoptotic calcium channel P2X7 in the glandular epithelium.

    PubMed

    Slater, Michael; Danieletto, Suzanne; Barden, Julian A

    2005-03-01

    In the current study, expression of the apoptotic calcium channel receptor P2X(7) and prostate-specific antigen (PSA) levels were studied in biopsy cores from 174 patients as well as 20 radical prostatectomy cases. In clinical biopsies, we have previously demonstrated that P2X(1 )and P2X(2) calcium channel receptors are absent from normal prostate epithelium that does not progress to prostate cancer within 5 years. In cases that did progress to prostate cancer however, P2X(1 )and P2X(2) labeling was observed in a stage-specific manner first in the nucleus, then the cytoplasm and finally on the apical epithelium, as prostate cancer developed. These markers were present up to 5 years before cancer was detectable by the usual morphological criteria (Gleason grading) as determined by H and E staining. In the current study, the apoptotic calcium channel receptor P2X(7) yielded similar results to that of P2X(1) and P2X(2). Using radical prostatectomy tissue sections as well as biopsies, these changes in calcium channel metabolism were noted throughout the prostate, indicating a field effect. This finding suggests that the presence of a prostate tumor could be detected without the need for direct sampling of tumor tissue, leading to detection of false negative cases missed by H or E stain. The reliability of PSA levels as a prognostic indicator has been questioned in recent years. In the current study, PSA levels were correlated with the P2X(7) labeling results. All patients who exhibited no P2X(7) labeling had a prostatic serum antigen (PSA) level of <2. Patients who exhibited stage-specific P2X(7) expression, and who later developed obvious prostate cancer as diagnosed by H and E stain, all had a PSA > 2. This finding suggests that increasing PSA may be an accurate indicator of cancer development.

  1. Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

    DTIC Science & Technology

    2011-10-01

    impact of deletion of connexins (Cx30/Cx43) in astrocytes on post-traumatic ATP release. In vivo bioluminescence imaging showed a significant reduction...the observation that ATP release and activation of P2X7 receptors drives the innate inflammatory response initiated by spinal cord injury. P2X7R...swelling. Prior studies have shown that excessive ATP release from peri-traumatic regions contributes to the inflammatory response to SCI by

  2. Pharmacological identification of P2X1, P2X4 and P2X7 nucleotide receptors in the smooth muscles of human umbilical cord and chorionic blood vessels.

    PubMed

    Valdecantos, P; Briones, R; Moya, P; Germain, A; Huidobro-Toro, J P

    2003-01-01

    To ascertain the role of extracellular adenosine 5'-triphosphate (ATP) receptors in human placenta circulation, we identified and pharmacologically characterized the P2X receptor population in its superficial vessels. Total RNA was extracted from segments of chorionic and umbilical arteries and veins of terminal placentae delivered by vaginal or Caesarian births. Polymerase chain reaction (PCR), followed by sequencing of the products, identified the presence of P2X 1, 4, 5, 6, and 7mRNAs in smooth muscle from chorionic and umbilical arteries and veins. Umbilical vessels proximal to the fetus expressed the same population of P2X subtypes, except for the P2X(5), but additionally expressed the P2X(2). Rings of chorionic vessels contracted upon addition of nucleotides and analogs with the following relative rank order of potencies in arteries and veins: alpha,beta-methyleneATP>beta,gamma-methyleneATP>PNP>ATP=diBzATP>2-MeSATP>ADP>AMP; in umbilical vessels alpha,beta-methyleneATP was at least 100-fold more potent than ATP. Nucleotide potency was less than that of PGF(2alpha) or endothelin-2, but had the same magnitude as serotonin. ATP-desensitized receptors evidenced cross desensitization to alpha,beta-methyleneATP, 2-MeSATP and diBzATP, effect not observed when desensitization was elicited by alpha,beta-methyleneATP, confirming the presence of various P2X receptor subtypes in the smooth muscles of these vessels. The vasocontractile efficacy of alpha,beta-methyleneATP was unaltered by endothelium removal, while that of ATP was significantly attenuated and those elicited by 2-MeSATP were blunted, indicating the presence of additional endothelial nucleotide receptors. These results suggest that P2X receptors participate in the humoral regulation of placental blood flow.

  3. 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.

  4. TRPM7 is a molecular substrate of ATP-evoked P2X7-like currents in tumor cells

    PubMed Central

    Nörenberg, Wolfgang; Plötz, Tanja; Sobottka, Helga; Chubanov, Vladimir; Mittermeier, Lorenz; Kalwa, Hermann; Aigner, Achim

    2016-01-01

    Within the ion channel–coupled purine receptor (P2X) family, P2X7 has gained particular interest because of its role in immune responses and in the growth control of several malignancies. Typical hallmarks of P2X7 are nonselective and noninactivating cation currents that are elicited by high concentrations (0.1–10 mM) of extracellular ATP. Here, we observe spurious ATP-induced currents in HEK293 cells that neither express P2X7 nor display ATP-induced Ca2+ influx or Yo-Pro-1 uptake. Although the biophysical properties of these ionic currents resemble those of P2X7 in terms of their reversal potential close to 0 mV, nonrectifying current-voltage relationship, current run-up during repeated ATP application, and augmentation in bath solutions containing low divalent cation (DIC) concentrations, they are poorly inhibited by established P2X7 antagonists. Because high ATP concentrations reduce the availability of DICs, these findings prompted us to ask whether other channel entities may become activated by our experimental regimen. Indeed, a bath solution with no added DICs yields similar currents and also a rapidly inactivating Na+-selective conductance. We provide evidence that TRPM7 and ASIC1a (acid-sensing ion channel type Ia)-like channels account for these noninactivating and phasic current components, respectively. Furthermore, we find ATP-induced currents in rat C6 glioma cells, which lack functional P2X receptors but express TRPM7. Thus, the observation of an atypical P2X7-like conductance may be caused by the activation of TRPM7 by ATP, which scavenges free DICs and thereby releases TRPM7 from permeation block. Because TRPM7 has a critical role in controlling the intracellular Mg2+ homeostasis and regulating tumor growth, these data imply that the proposed role of P2X7 in C6 glioma cell proliferation deserves reevaluation. PMID:27185858

  5. 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

  6. Activation of P2X7 promotes cerebral edema and neurological injury after traumatic brain injury in mice.

    PubMed

    Kimbler, Donald E; Shields, Jessica; Yanasak, Nathan; Vender, John R; Dhandapani, Krishnan M

    2012-01-01

    Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-1β (IL-1β) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, brilliant blue G (BBG), a clinically non-toxic P2X7 inhibitor, inhibited IL-1β expression, limited edemic development, and improved neurobehavioral outcomes after TBI. The beneficial effects of BBG followed either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and attenuated the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.

  7. Differential role of pannexin-1/ATP/P2X7 axis in IL-1β release by human monocytes.

    PubMed

    Parzych, Katarzyna; Zetterqvist, Anna V; Wright, William R; Kirkby, Nicholas S; Mitchell, Jane A; Paul-Clark, Mark J

    2017-02-28

    IL-1β release is integral to the innate immune system. The release of mature IL-1β depends on 2 regulated events: the de novo induction of pro-IL-1β, generally via NF-κB-dependent transduction pathways; and the assembly and activation of the NLRP3 inflammasome. This latter step is reliant on active caspase-1, pannexin-1, and P2X7 receptor activation. Pathogen-associated molecular patterns in gram-positive and gram-negative bacteria activate IL-1β release from immune cells via TLR2 and TLR4 receptors, respectively. We found that pro-IL-1β and mature IL-1β release from human monocytes is stimulated by the TLR2 agonists Pam3CSK4 or FSL-1 as well as the TLR4 agonist LPS in the absence of additional ATP. TLR2 agonists required pannexin-1 and P2X7 receptor activation to stimulate IL-1β release. In contrast, IL-1β release stimulated by the TLR4 agonist LPS is independent of both pannexin-1 and P2X7 activation. In the absence of exogenous ATP, P2X7 activation requires endogenous ATP release, which occurs in some cells via pannexin-1. In line with this, we found that LPS-stimulated human monocytes released relatively low levels of ATP, whereas cells stimulated with TLR2 agonists released high levels of ATP. These findings suggest that in human monocytes, both TLR2 and TLR4 signaling induce pro-IL-1β expression, but the mechanism by which they activate caspase-1 diverges at the level of the pannexin-1/ATP/P2X7 axis.-Parzych, K., Zetterqvist, A. V., Wright, W. R., Kirkby, N. S., Mitchell, J. A., Paul-Clark, M. J. Differential role of pannexin-1/ATP/P2X7 axis in IL-1β release by human monocytes.

  8. Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists

    PubMed Central

    Chaïb, N; Kabré, E; Alzola, E; Pochet, S; Dehaye, J P

    2000-01-01

    The permeabilizing effect of P2X7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37°C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 μM) and it was decreased when the temperature was lowered to 25°C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 μM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A2 (iPLA2). Bromoenol lactone (BEL) inhibited the iPLA2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X7 receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor. PMID:10683195

  9. Amplification loop of the inflammatory process is induced by P2X7R activation in intestinal epithelial cells in response to neutrophil transepithelial migration.

    PubMed

    Cesaro, Annabelle; Brest, Patrick; Hofman, Véronique; Hébuterne, Xavier; Wildman, Scott; Ferrua, Bernard; Marchetti, Sandrine; Doglio, Alain; Vouret-Craviari, Valérie; Galland, Franck; Naquet, Philippe; Mograbi, Baharia; Unwin, Robert; Hofman, Paul

    2010-07-01

    Inflammatory bowel diseases (IBD) are characterized during their active phase by polymorphonuclear leukocyte (PMNL) transepithelial migration. The efflux of PMNL into the mucosa is associated with the production of proinflammatory cytokines and the release of ATP from damaged and necrotic cells. The expression and function of purinergic P2X(7) receptor (P2X(7)R) in intestinal epithelial cells (IEC) and its potential role in the "cross talk" between IEC and PMNL have not been explored. The aims of the present study were 1) to examine P2X(7)R expression in IEC (T84 cells) and in human intestinal biopsies; 2) to detect any changes in P2X(7)R expression in T84 cells during PMNL transepithelial migration, and during the active and quiescent phases of IBD; and 3) to test whether P2X(7)R stimulation in T84 monolayers can induce caspase-1 activation and IL-1beta release by IEC. We found that a functional ATP-sensitive P2X(7)R is constitutively expressed at the apical surface of IEC T84 cells. PMNL transmigration regulates dynamically P2X(7)R expression and alters its distribution from the apical to basolateral surface of IEC during the early phase of PMNL transepithelial migration in vitro. P2X(7)R expression was weak in intestinal biopsies obtained during the active phase of IBD. We show that activation of epithelial P2X(7)R is mandatory for PMNL-induced caspase-1 activation and IL-1beta release by IEC. Overall, these changes in P2X(7)R function may serve to tailor the intensity of the inflammatory response and to prevent IL-1beta overproduction and inflammatory disease.

  10. Rhinovirus-Induced IL-1β Release from Bronchial Epithelial Cells Is Independent of Functional P2X7

    PubMed Central

    Shi, Lei; Manthei, David M.; Guadarrama, Arturo G.; Lenertz, Lisa Y.

    2012-01-01

    Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor–dependent inflammatory responses, largely through P2X7. Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X7 to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X7 messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X7 was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2′-3′-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro–IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X7 in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing. PMID:22493010

  11. Involvement of sodium in early phosphatidylserine exposure and phospholipid scrambling induced by P2X7 purinoceptor activation in thymocytes.

    PubMed

    Courageot, Marie-Pierre; Lépine, Sandrine; Hours, Michel; Giraud, Françoise; Sulpice, Jean-Claude

    2004-05-21

    Extracellular ATP (ATP(ec)), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP(ec) induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP(ec)-induced Ca(2+) and/or Na(+) influx. At physiological external Na(+) concentration, without external Ca(2+), PS was exposed in all ATP(ec)-responsive cells. In contrast, without external Na(+), physiological external Ca(2+) concentration promoted a submaximal response. Altogether these data show that Na(+) influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.

  12. Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

    DTIC Science & Technology

    2013-10-01

    microglia in response to SCI, and of the role of P2X7 receptor activation in that process . Indeed, in parallel work using uninjured human brain tissue...distinct glial fates are instructed, and how that process may be manipulated for therapeutic purposes. Thus, on the basis of the injury-associated...autocrine loop for the self -maintenance of glial progenitors, the perturbation of which might dictate progenitor recruitment as either reactive glia or

  13. Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

    DTIC Science & Technology

    2011-10-01

    evaluated the impact of deletion of connexins (Cx30/Cx43) in astrocytes on post-traumatic ATP release. In vivo bioluminescence imaging showed a...Introduction The proposed studies were based on the observati on that ATP release and activation of P2X7 receptors drives the innate inf lammatory...tissue swelling. Prior studies have shown that excess ive ATP release from peri-traumatic regions contributes to the inflammatory response to SCI by

  14. Glutathione induces GABA release through P2X7R activation on Müller glia.

    PubMed

    Freitas, Hércules Rezende; Reis, Ricardo A de Melo

    2017-01-01

    The retinal tissue of warm-blooded vertebrates performs surprisingly complex and accurate transduction of visual information. To achieve precision, a multilayered neuroglia structure is established throughout the embryonic development, and the presence of radial Müller (glial) cells ensure differentiation, growth and survival for the neuronal elements within retinal environment. It is assumed that Müller cells serve as a dynamic reservoir of progenitors, capable of expressing transcription factors, differentiating and proliferating as either neuronal or glial cells depending on extrinsic cues. In the postnatal period, Müller glia may re-enter cell cycle and produce new retinal neurons in response to acute damage. In this context, glutathione (GSH), a virtually ubiquitous tripeptide antioxidant, which is found at milimolar concentrations in central glial cells, plays a vital role as a reducing agent, buffering radical oxygen species (ROS) and preventing cell death in severely injured retinal tissues. Despite its antioxidant role, data also point to GSH as a signaling agent, suggesting that GABA release and P2X7R-mediated calcium inwards occur in Müller cells in a GSH-enriched environment. These phenomena indicate a novel mechanistic response to damage in the vertebrate retinal tissue, particularly in neuron-glia networks.

  15. Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis.

    PubMed

    Apolloni, Savina; Amadio, Susanna; Parisi, Chiara; Matteucci, Alessandra; Potenza, Rosa L; Armida, Monica; Popoli, Patrizia; D'Ambrosi, Nadia; Volonté, Cinzia

    2014-09-01

    In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a 'gene modifier' in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by

  16. Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Apolloni, Savina; Amadio, Susanna; Parisi, Chiara; Matteucci, Alessandra; Potenza, Rosa L.; Armida, Monica; Popoli, Patrizia; D’Ambrosi, Nadia; Volonté, Cinzia

    2014-01-01

    In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by

  17. 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.

  18. P2X7 antagonism using Brilliant Blue G reduces body weight loss and prolongs survival in female SOD1G93A amyotrophic lateral sclerosis mice

    PubMed Central

    Bartlett, Rachael; Sluyter, Vanessa; Watson, Debbie

    2017-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterised by the accumulation of aggregated proteins, microglia activation and motor neuron loss. The mechanisms underlying neurodegeneration and disease progression in ALS are unknown, but the ATP-gated P2X7 receptor channel is implicated in this disease. Therefore, the current study aimed to examine P2X7 in the context of neurodegeneration, and investigate whether the P2X7 antagonist, Brilliant Blue G (BBG), could alter disease progression in a murine model of ALS. Methods Human SOD1G93A transgenic mice, which normally develop ALS, were injected with BBG or saline, three times per week, from pre-onset of clinical disease (62–64 days of age) until end-stage. During the course of treatment mice were assessed for weight, clinical score and survival, and motor coordination, which was assessed by rotarod performance. Various parameters from end-stage mice were assessed as follows. Motor neuron loss and microgliosis were assessed by immunohistochemistry. Relative amounts of lumbar spinal cord SOD1 and P2X7 were quantified by immunoblotting. Serum monocyte chemoattractant protein-1 was measured by ELISA. Splenic leukocyte populations were assessed by flow cytometry. Relative expression of splenic and hepatic P2X7 mRNA was measured by quantitative real-time PCR. Lumbar spinal cord SOD1 and P2X7 were also quantified by immunoblotting in untreated female SOD1G93A mice during the course of disease. Results BBG treatment reduced body weight loss in SOD1G93A mice of combined sex, but had no effect on clinical score, survival or motor coordination. BBG treatment reduced body weight loss in female, but not male, SOD1G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1G93A mice, extending the mean survival time by 4.3% in female mice compared to female mice treated with saline. BBG treatment had no effect on clinical score or motor coordination in

  19. Contribution of the P2X7 1513A/C loss-of-function polymorphism to extrapulmonary tuberculosis susceptibility in Tunisian populations.

    PubMed

    Ben-Selma, Walid; Ben-Kahla, Imen; Boukadida, Jalel; Harizi, Hedi

    2011-10-01

    The P2X7 receptor has been found to be linked to an increased risk for tuberculosis in some populations. In this study, we investigate whether the P2X7 receptor plays a role in increasing susceptibility to tuberculosis in Tunisia. We examined two 1513A/C and -762T/C polymorphisms at the P2X7 receptor in 168 patients with pulmonary TB (pTB), 55 patients with extrapulmonary TB (epTB) and 150 blood donors from Tunisia. Genotyping of 1513A/C and -762T/C polymorphisms was performed in purified genomic DNA using PCR-restriction fragment length polymorphism and allele-specific PCR, respectively. The 1513C, CC and AC loss-of-function allele and genotypes were overrepresented in the epTB group compared with the control group (45% vs. 17%, P=10(-8) ; 24% vs. 4%, P=3 × 10(-7) ; 42% vs. 27%, P=10(-3) , respectively). Additionally, they were associated with 3.83-, 11.86- and 3.15-fold risks of developing this clinical tuberculosis form, respectively. No associations between the -762T/C polymorphism and tuberculosis disease, as well as disease anatomic location were observed. Collectively, our results suggest that the P2X7 1513A/C loss-of-function polymorphism may contribute to susceptibility to epTB in Tunisian populations.

  20. Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis.

    PubMed

    Chandrashekaran, Varun; Das, Suvarthi; Seth, Ratanesh Kumar; Dattaroy, Diptadip; Alhasson, Firas; Michelotti, Gregory; Nagarkatti, Mitzi; Nagarkatti, Prakash; Diehl, Anna Mae; Chatterjee, Saurabh

    2016-01-01

    Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused an increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression.

  1. 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.

  2. Involvement of the chemokine CCL3 and the purinoceptor P2X7 in the spinal cord in paclitaxel-induced mechanical allodynia

    PubMed Central

    2014-01-01

    Background Paclitaxel is an effective chemotherapeutic agent widely used for the treatment of solid tumors. The major dose-limiting toxicity of paclitaxel is peripheral neuropathy. The mechanisms underlying the development and maintenance of paclitaxel-induced peripheral neuropathy are still unclear, and there are no currently established effective treatments. Accumulating evidence in models of neuropathic pain in which peripheral nerves are lesioned has implicated spinal microglia and chemokines in pain hypersensitivity, but little is know about their roles in chemotherapy-induced peripheral neuropathy. In the present study, we investigated the role of CC-chemokine ligand 3 (CCL3) in the spinal cord in the development and maintenance of mechanical allodynia using a rat model of paclitaxel-induced neuropathy. Findings Repeated intravenous administration of paclitaxel induced a marked decrease in paw withdrawal threshold in response to mechanical stimulation (mechanical allodynia). In these rats, the number of microglia in the spinal dorsal horn (SDH) was significantly increased. Paclitaxel-treated rats showed a significant increase in the expression of mRNAs for CCL3 and its receptor CCR5 in the SDH. Intrathecal administration of a CCL3-neutralizing antibody not only attenuated the development of paclitaxel-induced mechanical allodynia but also reversed its maintenance. Paclitaxel also upregulated expression of purinoceptor P2X7 receptors (P2X7Rs), which have been implicated in the release of CCL3 from microglia, in the SDH. The selective P2X7R antagonist A438079 had preventive and reversal effects on paclitaxel-induced allodynia. Conclusions Our findings suggest a contribution of CCL3 and P2X7Rs in the SDH to paclitaxel-induced allodynia and may provide new therapeutic targets for paclitaxel-induced painful neuropathy. PMID:25127716

  3. 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

  4. 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.

  5. M1 and M2 Functional Imprinting of Primary Microglia: Role of P2X7 Activation and miR-125b

    PubMed Central

    Parisi, Chiara; Napoli, Giulia

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a most frequently occurring and severe form of motor neuron disease, causing death within 3–5 years from diagnosis and with a worldwide incidence of about 2 per 100,000 person-years. Mutations in over twenty genes associated with familial forms of ALS have provided insights into the mechanisms leading to motor neuron death. Moreover, mutations in two RNA binding proteins, TAR DNA binding protein 43 and fused in sarcoma, have raised the intriguing possibility that perturbations of RNA metabolism, including that of the small endogenous RNA molecules that repress target genes at the posttranscriptional level, that is, microRNAs, may contribute to disease pathogenesis. At present, the mechanisms by which microglia actively participate to both toxic and neuroprotective actions in ALS constitute an important matter of research. Among the pathways involved in ALS-altered microglia responses, in previous works we have uncovered the hyperactivation of P2X7 receptor by extracellular ATP and the overexpression of miR-125b, both leading to uncontrolled toxic M1 reactions. In order to shed further light on the complexity of these processes, in this short review we will describe the M1/M2 functional imprinting of primary microglia and a role played by P2X7 and miR-125b in ALS microglia activation. PMID:28090150

  6. Purinergic receptor X7 is a key modulator of metabolic oxidative stress-mediated autophagy and inflammation in experimental nonalcoholic steatohepatitis.

    PubMed

    Das, Suvarthi; Seth, Ratanesh Kumar; Kumar, Ashutosh; Kadiiska, Maria B; Michelotti, Gregory; Diehl, Anna Mae; Chatterjee, Saurabh

    2013-12-01

    Recent studies indicate that metabolic oxidative stress, autophagy, and inflammation are hallmarks of nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanisms that link these important events in NASH remain unclear. In this study, we investigated the mechanistic role of purinergic receptor X7 (P2X7) in modulating autophagy and resultant inflammation in NASH in response to metabolic oxidative stress. The study uses two rodent models of NASH. In one of them, a CYP2E1 substrate bromodichloromethane is used to induce metabolic oxidative stress and NASH. Methyl choline-deficient diet feeding is used for the other NASH model. CYP2E1 and P2X7 receptor gene-deleted mice are used to establish their roles in regulating metabolic oxidative stress and autophagy. Autophagy gene expression, protein levels, confocal microscopy based-immunolocalization of lysosome-associated membrane protein (LAMP)2A and histopathological analysis were performed. CYP2E1-dependent metabolic oxidative stress induced increases in P2X7 receptor expression and chaperone-mediated autophagy markers LAMP2A and heat shock cognate 70 but caused depletion of light chain 3 isoform B (LC3B) protein levels. P2X7 receptor gene deletion significantly decreased LAMP2A and inflammatory indicators while significantly increasing LC3B protein levels compared with wild-type mice treated with bromodichloromethane. P2X7 receptor-deleted mice were also protected from NASH pathology as evidenced by decreased inflammation and fibrosis. Our studies establish that P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, thereby modulating hepatic inflammation. Furthermore, our findings presented here form a basis for P2X7 receptor as a potential therapeutic target in the treatment for NASH.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Pathological concentrations of homocysteine increases IL-1β production in macrophages in a P2X7, NF-ĸB, and erk-dependent manner.

    PubMed

    Zanin, Rafael Fernandes; Bergamin, Letícia Scussel; Morrone, Fernanda Bueno; Coutinho-Silva, Robson; de Souza Wyse, Angela Terezinha; Battastini, Ana Maria Oliveira

    2015-12-01

    Elevated plasma levels of homocysteine (Hcy) are associated with the development of coronary artery disease (CAD), peripheral vascular disease, and atherosclerosis. Hyperhomocysteinemia is likely related to the enhanced production of pro-inflammatory cytokines including IL-1β. However, the mechanisms underlying the effects of Hcy in immune cells are not completely understood. Recent studies have established a link between macrophage accumulation, cytokine IL-1β, and the advance of vascular diseases. The purpose of the present study is to investigate the effects of Hcy on IL-1β secretion by murine macrophages. Hcy (100 μM) increases IL-1β synthesis via enhancement of P2X7 expression and NF-ĸB and ERK activation in murine macrophages. In addition, the antioxidant agent N-acetylcysteine (NAC) reduces NF-κB activation, ERK phosphorylation, and IL-1β production in Hcy-exposed macrophages, indicating the importance of ROS in this pro-inflammatory process. In summary, our results show that Hcy may be involved in the synthesis and secretion of IL-1β via NF-ĸB, ERK, and P2X7 stimulation in murine macrophages.

  12. Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells

    PubMed Central

    Salvestrini, Valentina; Orecchioni, Stefania; Talarico, Giovanna; Reggiani, Francesca; Mazzetti, Cristina; Bertolini, Francesco; Orioli, Elisa; Adinolfi, Elena; Virgilio, Francesco Di; Pezzi, Annalisa; Cavo, Michele

    2017-01-01

    Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2×7R is the most consistently expressed by tumors. P2×7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2×7R. Here, we show that P2×7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2×7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo. Overall, our results demonstrate that P2×7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML. PMID:27980223

  13. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

    PubMed Central

    Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2017-01-01

    ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury. PMID:28163685

  14. Toll-Like Receptors 2, -3 and -4 Prime Microglia but not Astrocytes Across Central Nervous System Regions for ATP-Dependent Interleukin-1β Release

    PubMed Central

    Facci, Laura; Barbierato, Massimo; Marinelli, Carla; Argentini, Carla; Skaper, Stephen D.; Giusti, Pietro

    2014-01-01

    Interleukin-1β (IL-1β) is a crucial mediator in the pathogenesis of inflammatory diseases at the periphery and in the central nervous system (CNS). Produced as an unprocessed and inactive pro-form which accumulates intracellularly, release of the processed cytokine is strongly promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Microglia are central to the inflammatory process and a major source of IL-1β when activated. Here we show that purified (>99%) microglia cultured from rat cortex, spinal cord and cerebellum respond robustly to ATP-dependent IL-1β release, upon priming with a number of TLR isoform ligands (zymosan and Pam3CSK4 for TLR2, poly(I:C) for TLR3). Cytokine release was prevented by a P2X7R antagonist and inhibitors of stress-activated protein kinases. Enriched astrocytes (≤5% microglia) from these CNS regions displayed responses qualitatively similar to microglia but became unresponsive upon eradication of residual microglia with the lysosomotropic agent Leu-Leu-OMe. Activation of multiple TLR isoforms in nervous system pathology, coupled with elevated extracellular ATP levels and subsequent P2X7R activation may represent an important route for microglia-derived IL-1β. This phenomenon may have important consequences for neuroinflammation and its position to the common pathology of CNS diseases. PMID:25351234

  15. Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

    PubMed Central

    Gilbert, Daniel F.; Stebbing, Martin J.; Kuenzel, Katharina; Murphy, Robyn M.; Zacharewicz, Evelyn; Buttgereit, Andreas; Stokes, Leanne; Adams, David J.; Friedrich, Oliver

    2016-01-01

    Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into

  16. 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.

  17. Receptor-Mediated Tobacco Toxicity

    PubMed Central

    Arredondo, Juan; Chernyavsky, Alexander I.; Marubio, Lisa M.; Beaudet, Arthur L.; Jolkovsky, David L.; Pinkerton, Kent E.; Grando, Sergei A.

    2005-01-01

    Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified α3β2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic

  18. Therapeutic Targeting of P2X7 after TBI

    DTIC Science & Technology

    2012-11-16

    evacuation of acute subdural hematoma : brain shift and the dynamics of extraaxial collections. Neurol Res, 26(7), 763-766. Cold, G. E., & Jensen, F. T...of consecutive bilateral surgeries for patients with acute 31 Kimbler, Donald E N10-P10 subdural hematoma who develop contralateral acute epi...or subdural hematoma . Surg Neurol, 60(1), 23-30; discussion 30. Miller, J. D., Becker, D. P., Ward, J. D., Sullivan, H. G., Adams, W. E., & Rosner, M

  19. Toll-like receptor 7 mediates pruritus.

    PubMed

    Liu, Tong; Xu, Zhen-Zhong; Park, Chul-Kyu; Berta, Temugin; Ji, Ru-Rong

    2010-12-01

    Toll-like receptors are typically expressed in immune cells to regulate innate immunity. We found that functional Toll-like receptor 7 (TLR7) was expressed in C-fiber primary sensory neurons and was important for inducing itch (pruritus), but was not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Our results indicate that TLR7 mediates itching and is a potential therapeutic target for anti-itch treatment in skin disease conditions.

  20. Receptor-mediated DNA-targeted photoimmunotherapy.

    PubMed

    Karagiannis, Tom C; Lobachevsky, Pavel N; Leung, Brenda K Y; White, Jonathan M; Martin, Roger F

    2006-11-01

    We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

  1. Receptor-mediated signaling in Aspergillus fumigatus

    PubMed Central

    Grice, C. M.; Bertuzzi, M.; Bignell, E. M.

    2013-01-01

    Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted. PMID:23430083

  2. Synaptic NMDA Receptors Mediate Hypoxic Excitotoxic Death

    PubMed Central

    Wroge, Christine M.; Hogins, Joshua; Eisenman, Larry; Mennerick, Steven

    2012-01-01

    Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDA receptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDAR hypothesis posits that synaptic NMDARs are neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. In part, the extrasynaptic hypothesis is built on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a non-substrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase (GPT), which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate. PMID:22573696

  3. Toll-like Receptor-7 Mediates Pruritus

    PubMed Central

    Liu, Tong; Xu, Zhen-Zhong; Park, Chul-Kyu; Berta, Temugin; Ji, Ru-Rong

    2010-01-01

    Toll-like receptors (TLRs) are typically expressed in immune cells to regulate innate immunity. Here we report that functional TLR7 is expressed in C-fiber primary sensory neurons and important for inducing itch (pruritis) but not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Thus, we have uncovered TLR7 as a novel itch mediator and a potential therapeutic target for anti-itch treatment in skin disease conditions. PMID:21037581

  4. The neural-glial purinergic receptor ensemble in chronic pain states.

    PubMed

    Jarvis, Michael F

    2010-01-01

    Chronic pain is characterized by enhanced sensory neurotransmission that underlies increased sensitivity to noxious stimuli and the perception of non-noxious stimuli as painful. Evidence from neurophysiological and pharmacological studies demonstrates that ATP produces pain by directly enhancing neuronal excitability via the activation of specific ligand-gated ion channels, the P2X3 and P2X2/3 receptors. In addition, ATP activates CNS glial cells (e.g. microglia) in response to persistent nociceptive stimulation. This latter effect involves several distinct receptor-mediated signaling pathways linked to the P2X4, P2X7 and P2Y(12) receptors. This review summarizes new data that places these purinergic signaling events in a mechanistic context that illustrates the ability of ATP to initiate and maintain states of heightened sensory neuron excitability associated with persistent pain.

  5. Receptors mediating toxicity and their involvement in endocrine disruption.

    PubMed

    Rüegg, Joëlle; Penttinen-Damdimopoulou, Pauliina; Mäkelä, Sari; Pongratz, Ingemar; Gustafsson, Jan-Ake

    2009-01-01

    Many toxic compounds exert their harmful effects by activating of certain receptors, which in turn leads to dysregulation of transcription. Some of these receptors are so called xenosensors. They are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. Other receptors that are modulated by toxic substances are hormone receptors, particularly the ones of the nuclear receptor family. Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Furthermore, excessive activation of the xenosensors can lead to disturbances of the integrity of the system as well. In this chapter, the concepts of receptor-mediated toxicity and hormone disruption are introduced. We start by describing environmental chemicals that can bind to xenosensors and nuclear hormone receptors. We then describe the receptors most commonly targeted by environmental chemicals. Finally, the mechanisms by which receptor-mediated events can disrupt the system are depicted.

  6. Ligand-gated purinergic receptors regulate HIV-1 Tat and morphine related neurotoxicity in primary mouse striatal neuron-glia co-cultures.

    PubMed

    Sorrell, Mary E; Hauser, Kurt F

    2014-03-01

    Emerging evidence suggests that opioid drugs, such as morphine and heroin, can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that opioid drugs acting via μ opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. Key aspects of microglial function are regulated by the P2X family of ATP activated ligand-gated ion channels. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, which prompted us to investigate whether the cation-permeable P2X receptors contribute to the neurotoxic effects of HIV and morphine. To address this question, neuron survival, as well as other endpoints including changes in dendritic length, extracellular ATP levels, and intracellular calcium levels, were assayed in primary neuron-glia co-cultures from mouse striatum. Treatment with TNP-ATP, a non-selective P2X antagonist, prevented the neurotoxic effects of exposure to morphine and/or HIV Tat, or ATP alone, suggesting P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. Although P2X7, and perhaps P2X1, receptor activation decreases neuron survival, neither P2X1, P2X3, nor P2X7 selective receptor antagonists prevented Tat and/or morphine-induced neurotoxicity. These and other experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and provide circumstantial evidence implicating P2X4 receptors in particular. Our findings reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompanies neuroAIDS and opiate abuse.

  7. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  8. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  9. 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.

  10. NMDA Receptors Mediate Synaptic Competition in Culture

    PubMed Central

    She, Kevin; Craig, Ann Marie

    2011-01-01

    Background Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. Methodology/Principal Findings GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. Conclusions/Significance The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous

  11. 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.

  12. 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.

  13. 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.

  14. Extracellular purines, purinergic receptors and tumor growth

    PubMed Central

    Di Virgilio, F; Adinolfi, E

    2017-01-01

    Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field. PMID:27321181

  15. Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

    PubMed

    Gicquel, Thomas; Victoni, Tatiana; Fautrel, Alain; Robert, Sacha; Gleonnec, Florence; Guezingar, Marie; Couillin, Isabelle; Catros, Véronique; Boichot, Elisabeth; Lagente, Vincent

    2014-04-01

    Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury.

  16. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    PubMed Central

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  17. 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. PMID:27635169

  18. The receptors that mediate the direct lethality of anthrax toxin.

    PubMed

    Liu, Shihui; Zhang, Yi; Hoover, Benjamin; Leppla, Stephen H

    2012-12-27

    Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8(-/-) and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2(-/-) mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2(-/-)/TEM8(-/-) mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2(-/-)/TEM8(-/-) mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.

  19. Toll Like Receptor-9 Mediated Invasion in Breast Cancer

    DTIC Science & Technology

    2012-07-01

    breast cancers. A likely mechanism of this clinical finding involves dif- ferential responses to hypoxia. Our pre-clinical studies indicate that while...outcome of TLR activation is an innate immune reaction characterized by increased production of inflammatory mediators [2]. Recent studies indicate that...A 14. ABSTRACT TLR9 is a cellular DNA-receptor that is widely expressed in breast cancers. The aim of this work was to study whether DNA

  20. Nicotinic and opioid receptor regulation of striatal dopamine D2-receptor mediated transmission

    PubMed Central

    Mamaligas, Aphroditi A.; Cai, Yuan; Ford, Christopher P.

    2016-01-01

    In addition to dopamine neuron firing, cholinergic interneurons (ChIs) regulate dopamine release in the striatum via presynaptic nicotinic receptors (nAChRs) on dopamine axon terminals. Synchronous activity of ChIs is necessary to evoke dopamine release through this pathway. The frequency-dependence of disynaptic nicotinic modulation has led to the hypothesis that nAChRs act as a high-pass filter in the dopaminergic microcircuit. Here, we used optogenetics to selectively stimulate either ChIs or dopamine terminals directly in the striatum. To measure the functional consequence of dopamine release, D2-receptor synaptic activity was assessed via virally overexpressed potassium channels (GIRK2) in medium spiny neurons (MSNs). We found that nicotinic-mediated dopamine release was blunted at higher frequencies because nAChRs exhibit prolonged desensitization after a single pulse of synchronous ChI activity. However, when dopamine neurons alone were stimulated, nAChRs had no effect at any frequency. We further assessed how opioid receptors modulate these two mechanisms of release. Bath application of the κ opioid receptor agonist U69593 decreased D2-receptor activation through both pathways, whereas the μ opioid receptor agonist DAMGO decreased D2-receptor activity only as a result of cholinergic-mediated dopamine release. Thus the release of dopamine can be independently modulated when driven by either dopamine neurons or cholinergic interneurons. PMID:27886263

  1. A classification of opiate receptors that mediate antinociception in animals.

    PubMed Central

    Tyers, M. B.

    1980-01-01

    1 To investigate the opiate receptors that mediate antinociception, the activity profiles of opioid analgesic drugs have been determined against different nociceptive stimuli in the mouse and rat. 2 In tests that employ heat as the nociceptive stimulus, mu-opiate receptor agonists, such as morphine, pethidine and dextropropoxyphene, had steep and parallel dose-response curves and were capable of achieving maximum effects. In addition, the antinociceptive potency ratios of these drugs in heat tests were similar to those for analgesia in man. 3 The kappa-agonists, such as ethylketazocine, nalorphine, Mr2034 and pentazocine, were essentially inactive against heat nociception except at doses that caused sedation and motor incapacitation. 4 In the writhing and paw pressure tests both mu- and kappa-agonists produced steep and parallel dose-response curves. 5 It is concluded that both mu- and kappa-opiate receptors mediate antinociception in animals and that the interactions of analgesic drugs with these receptors may be classified in terms of their antinociceptive activities against qualitatively different nociceptive stimuli. PMID:6249436

  2. Receptor-mediated choreography of life and death.

    PubMed

    Bhardwaj, Anjana; Aggarwal, Bharat B

    2003-09-01

    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  3. 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.

  4. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    PubMed Central

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  5. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    PubMed

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  6. 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

  7. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  8. Receptor- and reactive intermediate-mediated mechanisms of teratogenesis.

    PubMed

    Wells, Peter G; Lee, Crystal J J; McCallum, Gordon P; Perstin, Julia; Harper, Patricia A

    2010-01-01

    Drugs and environmental chemicals can adversely alter the development of the fetus at critical periods during pregnancy, resulting in death, or in structural and functional birth defects in the surviving offspring. This process of teratogenesis may not be evident until a decade or more after birth. Postnatal functional abnormalities include deficits in brain function, a variety of metabolic diseases, and cancer. Due to the high degree of fetal cellular division and differentiation, and to differences from the adult in many biochemical pathways, the fetus is highly susceptible to teratogens, typically at low exposure levels that do not harm the mother. Insights into the mechanisms of teratogenesis come primarily from animal models and in vitro systems, and involve either receptor-mediated or reactive intermediate-mediated processes. Receptor-mediated mechanisms involving the reversible binding of xenobiotic substrates to a specific receptor are exemplified herein by the interaction of the environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or "dioxin") with the cytosolic aryl hydrocarbon receptor (AHR), which translocates to the nucleus and, in association with other proteins, binds to AH-responsive elements (AHREs) in numerous genes, initiating changes in gene transcription that can perturb development. Alternatively, many xenobiotics are bioactivated by fetal enzymes like the cytochromes P450 (CYPs) and prostaglandin H synthases (PHSs) to highly unstable electrophilic or free radical reactive intermediates. Electrophilic reactive intermediates can covalently (irreversibly) bind to and alter the function of essential cellular macromolecules (proteins, DNA), causing developmental anomalies. Free radical reactive intermediates can enhance the formation of reactive oxygen species (ROS), resulting in oxidative damage to cellular macromolecules and/or altered signal transduction. The teratogenicity of reactive intermediates is determined to a large extent

  9. Opioid receptor internalization contributes to dermorphin-mediated antinociception

    PubMed Central

    Macey, Tara A.; Ingram, Susan L.; Bobeck, Erin N.; Hegarty, Deborah M.; Aicher, Sue A.; Arttamangkul, Seksiri; Morgan, Michael M.

    2010-01-01

    Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-CNA prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABAA antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization. PMID:20394808

  10. Menthol inhibits 5-HT3 receptor-mediated currents.

    PubMed

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  11. 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

  12. Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.

    PubMed

    Lopez, Joëlle; Gamache, Karine; Schneider, Rilla; Nader, Karim

    2015-02-11

    Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

  13. The brain hypocretins and their receptors: mediators of allostatic arousal.

    PubMed

    Carter, Matthew E; Schaich Borg, Jana; de Lecea, Luis

    2009-02-01

    The hypocretins (abbreviated 'Hcrts' - also called 'orexins') are two neuropeptides secreted exclusively by a small population of neurons in the lateral hypothalamus. These peptides bind to two receptors located throughout the brain in nuclei associated with diverse cognitive and physiological functions. Initially, the brain Hcrt system was found to have a major role in the regulation of sleep/wake transitions. More recent studies indicate Hcrts may play a role in other physiological functions, including food intake, addiction, and stress. Taken together, these studies suggest a general role for Hcrts in mediating arousal, especially when an organism must respond to unexpected stressors and challenges in the environment.

  14. Origin of Toll-like receptor-mediated innate immunity.

    PubMed

    Kanzok, Stefan M; Hoa, Ngo T; Bonizzoni, Mariangela; Luna, Coralia; Huang, Yaming; Malacrida, Anna R; Zheng, Liangbiao

    2004-04-01

    Toll-related receptors (TLR) have been found in four animal phyla: Nematoda, Arthropoda, Echinodermata, and Chordata. No TLR has been identified thus far in acoelomates. TLR genes play a pivotal role in the innate immunity in both fruit fly and mammals. The prevailing view is that TLR-mediated immunity is ancient. The two pseudocoelomate TLRs, one each from Caenorhabditis elegans and Strongyloides stercoralis, were distinct from the coelomate ones. Further, the only TLR gene (Tol-1) in Ca. elegans did not appear to play a role in innate immunity. We argue that TLR-mediated innate immunity developed only in the coelomates, after they split from pseudocoelomates and acoelomates. We hypothesize that the function of TLR-mediated immunity is to prevent microbial infection in the body cavity present only in the coelomates. Phylogenetic analysis showed that almost all arthropod TLRs form a separate cluster from the mammalian counterparts. We further hypothesize that TLR-mediated immunity developed independently in the protostomia and deuterostomia coelomates.

  15. Drosophila Ryanodine Receptors Mediate General Anesthesia by Halothane

    PubMed Central

    Gao, Shuying; Sandstrom, David J.; Smith, Harold E.; High, Brigit; Marsh, Jon W.; Nash, Howard A.

    2012-01-01

    Background Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and we hypothesized that it has a central role in anesthesia. Methods Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca2+ imaging and patch clamp electrophysiology. Results Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca2+ efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca2+ imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca2+ flux that is altered in dRyr mutants and correlates with strong hyperpolarization. Conclusions In Drosophila, neurally-expressed dRyr mediates a substantial proportion of halothane's anesthetic effects in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. Our results provide support for Ryr as an important mediator of immobilization by volatile anesthetics. PMID:23254148

  16. Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation.

    PubMed

    Dupakuntla, Madhavi; Pathak, Bhakti; Roy, Binita Sur; Mahale, Smita D

    2012-10-15

    The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling.

  17. CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy

    PubMed Central

    2014-01-01

    Background Painful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN. Results We demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice. Conclusions These data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN. PMID:24961298

  18. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

    PubMed

    Kalmanzon, Eliahu; Rahamim, Yocheved; Barenholz, Yechezkel; Carmeli, Shmuel; Zlotkin, Eliahu

    2003-07-01

    Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

  19. Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity.

    PubMed

    Wu, Fenfang; Chen, Liyong; Liu, Xin; Wang, Huaying; Su, Peng; Han, Yinglun; Feng, Bo; Qiao, Xu; Zhao, Jing; Ma, Ning; Liu, Huijie; Zheng, Zhen; Li, Qingwei

    2013-02-01

    An alternative adaptive-immune system is present in the most basal vertebrates--lampreys and hagfish--the only surviving jawless vertebrates. These eel-like fish use leucine-rich repeat-based receptors for Ag recognition instead of the Ig-based receptors used in jawed vertebrates. We report that in Japanese lamprey (Lampetra japonica), variable lymphocyte receptor (VLR)B interacts with C1q and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune-based lysis involves deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive-immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based Abs and the classical complement-activation pathway.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Dynamic kisspeptin receptor trafficking modulates kisspeptin-mediated calcium signaling.

    PubMed

    Min, Le; Soltis, Kathleen; Reis, Ana Claudia S; Xu, Shuyun; Kuohung, Wendy; Jain, Manisha; Carroll, Rona S; Kaiser, Ursula B

    2014-01-01

    Kisspeptin receptor (KISS1R) signaling plays a critical role in the regulation of reproduction. We investigated the role of kisspeptin-stimulated KISS1R internalization, recycling, and degradation in the modulation of KISS1R signaling. Kisspeptin stimulation of Chinese hamster ovary or GT1-7 cells expressing KISS1R resulted in a biphasic increase in intracellular Ca(2+) ([Ca(2+)]i), with a rapid acute increase followed by a more sustained second phase. In contrast, stimulation of the TRH receptor, another Gq/11-coupled receptor, resulted in a much smaller second-phase [Ca(2+)]i response. The KISS1R-mediated second-phase [Ca(2+)]i response was abolished by removal of kisspeptin from cell culture medium. Notably, the second-phase [Ca(2+)]i response was also inhibited by dynasore, brefeldin A, and phenylarsine oxide, which inhibit receptor internalization and recycling, suggesting that KISS1R trafficking contributes to the sustained [Ca(2+)]i response. We further demonstrated that KISS1R undergoes dynamic ligand-dependent and -independent recycling. We next investigated the fate of the internalized kisspeptin-KISS1R complex. Most internalized kisspeptin was released extracellularly in degraded form within 1 hour, suggesting rapid processing of the internalized kisspeptin-KISS1R complex. Using a biotinylation assay, we demonstrated that degradation of cell surface KISS1R was much slower than that of the internalized ligand, suggesting dissociated processing of the internalized kisspeptin-KISS1R complex. Taken together, our results suggest that the sustained calcium response to kisspeptin is dependent on the continued presence of extracellular ligand and is the result of dynamic KISS1R trafficking.

  5. Ligand Receptor-Mediated Regulation of Growth in Plants.

    PubMed

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  6. Nociceptive transmission and modulation via P2X receptors in central pain syndrome.

    PubMed

    Kuan, Yung-Hui; Shyu, Bai-Chuang

    2016-05-26

    Painful sensations are some of the most frequent complaints of patients who are admitted to local medical clinics. Persistent pain varies according to its causes, often resulting from local tissue damage or inflammation. Central somatosensory pathway lesions that are not adequately relieved can consequently cause central pain syndrome or central neuropathic pain. Research on the molecular mechanisms that underlie this pathogenesis is important for treating such pain. To date, evidence suggests the involvement of ion channels, including adenosine triphosphate (ATP)-gated cation channel P2X receptors, in central nervous system pain transmission and persistent modulation upon and following the occurrence of neuropathic pain. Several P2X receptor subtypes, including P2X2, P2X3, P2X4, and P2X7, have been shown to play diverse roles in the pathogenesis of central pain including the mediation of fast transmission in the peripheral nervous system and modulation of neuronal activity in the central nervous system. This review article highlights the role of the P2X family of ATP receptors in the pathogenesis of central neuropathic pain and pain transmission. We discuss basic research that may be translated to clinical application, suggesting that P2X receptors may be treatment targets for central pain syndrome.

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

    PubMed

    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 Ca(2+) (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 Ca(2+) 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.

  8. 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.

  9. Herpes virus entry mediator synergizes with Toll-like receptor mediated neutrophil inflammatory responses

    PubMed Central

    Haselmayer, Philipp; Tenzer, Stefan; Kwon, Byoung S; Jung, Gundram; Schild, Hansjörg; Radsak, Markus P

    2006-01-01

    In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the function of HVEM on PMN, we evaluated the effect of receptor ligation on human PMN effector functions using an agonistic monoclonal antibody. Here we demonstrate that activation of HVEM causes activation of neutrophil effector functions, including respiratory burst, degranulation and release of interleukin-8 in synergy with ligands for Toll-like receptors or GM-CSF. In addition, stimulation via HVEM enhanced neutrophil phagocytic activity of complement opsonized, but not of non-opsonized, particles. In conclusion, these results indicate a new, as yet unknown, participation of HVEM in the innate immune response and points to a new link between innate and adaptive immunity. PMID:17067315

  10. Brain prostanoid TP receptor-mediated adrenal noradrenaline secretion and EP3 receptor-mediated sympathetic noradrenaline release in rats.

    PubMed

    Yokotani, Keiko; Okada, Shoshiro; Nakamura, Kumiko; Yamaguchi-Shima, Naoko; Shimizu, Takahiro; Arai, Junichi; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2005-04-04

    Sympathetic nerves release noradrenaline, whereas adrenal medullary chromaffin cells secrete noradrenaline and adrenaline. Therefore, plasma noradrenaline reflects the secretion from adrenal medulla in addition to the release from sympathetic nerves, however the exact mechanisms of adrenal noradrenaline secretion remain to be elucidated. The present study was designated to characterize the source of plasma noradrenaline induced by intracerebroventricularly (i.c.v.) administered bombesin and prostaglandin E2 in urethane-anesthetized rats. Bombesin (1.0 nmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline, while prostaglandin E2 (0.3 nmol/animal, i.c.v.) elevated only plasma noradrenaline. The bombesin-induced elevations of both catecholamines were attenuated by pretreatments with furegrelate (an inhibitor of thromboxane A2 synthase) [250 and 500 microg (0.9 and 1.8 micromol)/animal, i.c.v.)] and [(+)-S-145] [(+)-(1R,2R,3S,4S)-(5Z)-7-(3-[4-3H]-phenylsulphonyl-aminobicyclo[2.2.1]hept-2-yl)hept-5-enoic acid sodium salt] (an antagonist of prostanoid TP receptors) [100 and 250 microg (250 and 625 nmol)/animal)], and abolished by acute bilateral adrenalectomy. On the other hand, the prostaglandin E2-induced elevation of plasma noradrenaline was not influenced by acute bilateral adrenalectomy. These results suggest that adrenal noradrenaline secretion and sympathetic noradrenaline release are mediated by differential central mechanisms; brain prostanoid TP receptors activated by bombesin are involved in the adrenal noradrenaline secretion, while brain prostanoid EP (probably EP3) receptors activated by prostaglandin E2 are involved in the sympathetic noradrenaline release in rats. Brain prostanoid TP receptors activated by bombesin are also involved in the adrenal adrenaline secretion.

  11. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

    PubMed

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael

    2011-10-01

    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

  12. Retromer-Mediated Trafficking of Transmembrane Receptors and Transporters

    PubMed Central

    Klinger, Stine C.; Siupka, Piotr; Nielsen, Morten S.

    2015-01-01

    Transport between the endoplasmatic reticulum, the Golgi-network, the endo-lysosomal system and the cell surface can be categorized as anterograde or retrograde, describing traffic that goes forward or backward, respectively. Traffic going from the plasma membrane to endosomes and lysosomes or the trans-Golgi network (TGN) constitutes the major retrograde transport routes. Several transmembrane proteins undergo retrograde transport as part of a recycling mechanism that contributes to reutilization and maintenance of a steady-state protein localization. In addition, some receptors are hijacked by exotoxins and used for entry and intracellular transport. The physiological relevance of retrograde transport cannot be overstated. Retrograde trafficking of the amyloid precursor protein determines the distribution between organelles, and hence the possibility of cleavage by γ-secretase. Right balancing of the pathways is critical for protection against Alzheimer’s disease. During embryonic development, retrograde transport of Wntless to the TGN is essential for the following release of Wnt from the plasma membrane. Furthermore, overexpression of Wntless has been linked to oncogenesis. Here, we review relevant aspects of the retrograde trafficking of mammalian transmembrane receptors and transporters, with focus on the retromer-mediated transport between endosomes and the TGN. PMID:26154780

  13. Peripheral inflammation upregulates P2X receptor expression in satellite glial cells of mouse trigeminal ganglia: a calcium imaging study.

    PubMed

    Kushnir, Raya; Cherkas, Pavel S; Hanani, Menachem

    2011-09-01

    Satellite glial cells (SGCs) in sensory ganglia are altered structurally and biochemically as a result of nerve injury. Whereas there is ample evidence that P2 purinergic receptors in central glial cells are altered after injury, there is very little information on similar changes in SGCs, although it is well established that SGCs are endowed with P2 receptors. Using calcium imaging, we characterized changes in P2 receptors in SGCs from mouse trigeminal ganglia in short-term cultures. Seven days after the induction of submandibular inflammation with complete Freund's adjuvant, there was a marked increase in the sensitivity of SGCs to ATP, with the threshold of activation decreasing from 5 μM to 10 nM. A similar observation was made in the intact trigeminal ganglion after infra-orbital nerve axotomy. Using pharmacological tools, we investigated the receptor mechanisms underlying these changes in cultured SGCs. We found that in control tissues response to ATP was mediated by P2Y (metabotropic) receptors, whereas after inflammation the response was mediated predominantly by P2X (ionotropic) receptors. As the contribution of P2X1,3,6 receptors was excluded, and the sensitivity to a P2X7 agonist did not change after inflammation, it appears that after inflammation the responses to ATP are largely due to P2X2 and/or 5 receptors, with a possible contribution of P2X4 receptors. We conclude that inflammation induced a large increase in the sensitivity of SGCs to ATP, which involved a switch from P2Y to P2X receptors. We propose that the over 100-fold augmented sensitivity of SGCs to ATP after injury may contribute to chronic pain states.

  14. Scavenger receptor mediates systemic RNA interference in ticks.

    PubMed

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Xuenan, Xuan; Suzuki, Hiroshi; Galay, Remil Linggatong; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-01-01

    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.

  15. Scavenger Receptor Mediates Systemic RNA Interference in Ticks

    PubMed Central

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Xuenan, Xuan; Suzuki, Hiroshi; Linggatong Galay, Remil; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-01-01

    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks. PMID:22145043

  16. Rapid and estrogen receptor beta mediated actions in the hippocampus mediate some functional effects of estrogen

    PubMed Central

    Walf, Alicia A.; Frye, Cheryl A.

    2008-01-01

    The steroid hormone, estradiol (E2), has numerous targets in the central nervous system, including the hippocampus, which plays a key role in cognition and affective behavior. This review focuses on our evidence from studies in rodents that E2 has diverse mechanisms in the hippocampus for its functional effects E2 has rapid, membrane-mediated effects in the hippocampus to enhance cognitive performance. Administration of E2 to the hippocampus of rats for 10 minutes following training enhances performance in a hippocampus-mediated task. Increased cell firing in the hippocampus occurs within this short time frame. Furthermore, administration of free E2 or an E2 conjugate, E2:bovine serum albumin (BSA), to the hippocampus produces similar performance-enhancing effects in this task, suggesting that E2 has membrane actions in the hippocampus for these effects. Further evidence that E2 has rapid, membrane-mediated effects is that co-administration of E2 and inhibitors of mitogen activated protein kinase (MAPK), rather than intracellular E2 receptors (ERs) or protein synthesis, attenuate the enhancing effects of E2 in this task. Despite these data that demonstrate E2 can have rapid and/or membrane-mediated effects in the hippocampus, there is clear evidence to suggest that intracellular ERs, particularly the β (rather than α) isoform of ERs, may be important targets for E2’s functional effects for hippocampal processes. Administration of ligands that are specific for ERβ, but not ERα, have enhancing effects on hippocampal processes similar to that of E2 (which has similar affinity for ERα and ERβ). These effects are attenuated when ERβ expression is knocked down in transgenic models or with central administration of antisense oligonucleotides. Thus, there may be a convergence of E2’s actions through rapid, membrane-mediated effects and intracellular ERs and in the hippocampus for these functional effects. PMID:18342348

  17. 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...

  18. Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

    PubMed Central

    Olaru, Florina; Luo, Wentian; Suleiman, Hani; St. John, Patricia L.; Ge, Linna; Mezo, Adam R.; Shaw, Andrey S.; Abrahamson, Dale R.; Miner, Jeffrey H.

    2014-01-01

    The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG. PMID:24357670

  19. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    SciTech Connect

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D'Avis, J.C.; Wartofsky, L. )

    1991-03-01

    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  20. Current knowledge on the role of P2Y receptors in cardioprotection against ischemia-reperfusion.

    PubMed

    Djerada, Zoubir; Feliu, Catherine; Richard, Vincent; Millart, Hervé

    2017-04-01

    During ischemia, numerous effective endogenous extracellular mediators have been identified, particularly, nucleosides such as adenosine as well as purinergic and pyrimidinergic nucleotides. They may play important regulatory roles within the cardiovascular system and notably as cardio-protectants. Indeed, the distribution of the P2Y receptors in mammalian heart includes several cellular constituents relevant for the pathophysiology of myocardial ischemia. Beside the well-known cardioprotective effect of adenosine, the additional protective role of P2Y receptors has emerged. However, interpretation of experimental results may be sometimes perplexing. This is due to the variability of: the experimental models, the endpoints criteria, the chemical structure of agonist and antagonist ligands and their concentrations, the sequences of drug administration with respect to the model used (before and/or during and/or after ischemia). The net effect may be in the opposite direction after a transient or a prolonged stimulation. Nevertheless, the overall reading of published data highlights the beneficial role of the P2Y2/4 receptor stimulation, the useful and synergistic role of P2Y6/11 receptor activation and even of the P2Y11 receptor alone in cardioprotection. More, the P2Y11 receptor could be involved in counter-regulation of profibrotic processes. Paradoxically, transient P2X7 receptor stimulation could contribute to the net cardioprotective effect of ATP. Recently, experimental data have shown that blocking the P2Y12 receptor after ischemia confers cardioprotection independently of platelet antiaggregatory effect. This suggests for P2Y receptors an important role in primary prevention and as a therapeutic target in myocardial protection during ischemia and reperfusion.

  1. Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

    PubMed

    Gastel, J A; Sutter, T R

    1995-12-01

    We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

  2. Ryanodine receptor-2 upregulation and nicotine-mediated plasticity.

    PubMed

    Ziviani, Elena; Lippi, Giordano; Bano, Daniele; Munarriz, Eliana; Guiducci, Stefania; Zoli, Michele; Young, Kenneth W; Nicotera, Pierluigi

    2011-01-05

    Nicotine, the major psychoactive component of cigarette smoke, modulates neuronal activity to produce Ca2+-dependent changes in gene transcription. However, the downstream targets that underlie the long-term effects of nicotine on neuronal function, and hence behaviour, remain to be elucidated. Here, we demonstrate that nicotine administration to mice upregulates levels of the type 2 ryanodine receptor (RyR2), a Ca2+-release channel present on the endoplasmic reticulum, in a number of brain areas associated with cognition and addiction, notably the cortex and ventral midbrain. Nicotine-mediated RyR2 upregulation was driven by CREB, and caused a long-lasting reinforcement of Ca2+ signalling via the process of Ca2+-induced Ca2+ release. RyR2 upregulation was itself required for long-term phosphorylation of CREB in a positive-feedback signalling loop. We further demonstrate that inhibition of RyR-activation in vivo abolishes sensitization to nicotine-induced habituated locomotion, a well-characterised model for onset of drug dependence. Our findings, therefore, indicate that gene-dependent reprogramming of Ca2+ signalling is involved in nicotine-induced behavioural changes.

  3. Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

    PubMed

    Qian, Yue-Wei; Schmidt, Robert J; Zhang, Youyan; Chu, Shaoyou; Lin, Aimin; Wang, He; Wang, Xiliang; Beyer, Thomas P; Bensch, William R; Li, Weiming; Ehsani, Mariam E; Lu, Deshun; Konrad, Robert J; Eacho, Patrick I; Moller, David E; Karathanasis, Sotirios K; Cao, Guoqing

    2007-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.

  4. Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

    PubMed Central

    Taylor, A M W; Roberts, K W; Pradhan, A A; Akbari, H A; Walwyn, W; Lutfy, K; Carroll, F I; Cahill, C M; Evans, C J

    2015-01-01

    BACKGROUND AND PURPOSE The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception. EXPERIMENTAL APPROACH We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception. KEY RESULTS Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg−1), unmasked etorphine (3 mg·kg−1) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg−1) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg−1) and diazepam (1 mg·kg−1). CONCLUSIONS AND IMPLICATIONS Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles

  5. 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

  6. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    PubMed

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  7. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis

    PubMed Central

    Gonçalves-Carneiro, Daniel; McKeating, Jane A.

    2017-01-01

    ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between

  8. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    PubMed

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  9. 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

  10. NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors.

    PubMed

    Zhou, X; Hollern, D; Liao, J; Andrechek, E; Wang, H

    2013-03-28

    N-methyl-D-aspartate receptors (NMDAR) overactivation is linked to neurodegeneration. The current prevailing theory suggests that synaptic and extrasynaptic NMDAR (syn- and ex-NMDAR) impose counteracting effects on cell fate, and neuronal cell death is mainly mediated by the activation of ex-NMDAR. However, several lines of evidence implicate the limitation of this theory. Here, we demonstrate that activation of NMDAR bi-directionally regulated cell fate through stimulating pro-survival or pro-death signaling. While low-dose NMDA preferentially activated syn-NMDAR and stimulated the extracellular signal-regulated kinase ½-cAMP responsive element-binding protein-brain-derived neurotrophic factor pro-survival signaling, higher doses progressively activated increasing amount of ex-NMDAR along with syn-NMDAR and triggered cell death program. Interestingly, the activation of syn- or ex-NMDAR alone did not cause measurable cell death. Consistently, activation of syn- or ex-NMDAR alone stimulated pro-survival but not pro-death signaling. Next, we found that memantine, which was previously identified as an ex-NMDAR blocker, inhibited intracellular signaling mediated by syn- or ex-NMDAR. Simultaneous blockade of syn- and ex-NMDAR by memantine dose-dependently attenuated NMDAR-mediated death. Moreover, long- but not short-term treatment with high-dose NMDA or oxygen-glucose deprivation triggered cell death and suppressed pro-survival signaling. These data implicate that activation of syn- or ex-NMDAR alone is not neurotoxic. The degree of excitotoxicity depends on the magnitude and duration of syn- and ex-NMDAR coactivation. Finally, genome-wide examination demonstrated that the activation of syn- and ex-NMDAR lead to significant overlapping rather than counteracting transcriptional responses.

  11. Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

    PubMed Central

    Dozmorov, Mikhail; Li, Rui; Xu, Hui-Ping; Jilderos, Barbro; Wigström, Holger

    2004-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through

  12. GABAA Receptor-Mediated Activity in a Model of Cortical Dysplasia

    DTIC Science & Technology

    2012-06-29

    a model of cortical dysplasia" Name of Candidate: Joseph Abbah Doctor of Philosophy Degree June 29, 2012 DISSERTATION AND ABSTRACT APPROVED...receptor-mediated activity in a model of cortical dysplasia’ is appropriately acknowledged and, beyond brief excerpts, is with the permission of the...certifies that the use of any copyrighted material in this dissertation manuscript entitled: “GABAA receptor-mediated activity in a model of

  13. SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.

    PubMed Central

    Chen, J D; Umesono, K; Evans, R M

    1996-01-01

    Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8755515

  14. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    PubMed

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  15. 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…

  16. 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...

  17. 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

  18. Crosstalk Between Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2011-04-01

    the connection between obesity and breast cancer (5). Leptin , a product of the obese (ob) gene, is an adipocytokine that regulates appetite , bone... Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance Dr. Rita Nahta Emory University Atlanta, GA 30322 Obesity...hormones IGF-I and leptin and their receptors, IGF-IR and leptin receptor (Ob-R), are elevated in breast cancer. Co-immunoprecipitation and

  19. 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 ...

  20. 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...

  1. 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...

  2. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  3. Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

    PubMed

    Dobson, K S; Dmetrichuk, J M; Spencer, G E

    2006-09-15

    Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

  4. 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.

  5. 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

  6. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  7. Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization.

    PubMed

    Youn, Hyung S; Lee, Jun K; Choi, Yong J; Saitoh, Shin I; Miyake, Kensuke; Hwang, Daniel H; Lee, Joo Y

    2008-01-15

    Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFkappaB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFkappaB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFkappaB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNbeta in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFkappaB or IRF3 induced by MyD88-dependent (MyD88, IKKbeta) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFkappaB activation. Further, cinnamaldehyde inhibited ligand-independent NFkappaB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.

  8. Cannabinoid Receptor Activation Modifies NMDA Receptor Mediated Release of Intracellular Calcium: Implications for Endocannabinoid Control of Hippocampal Neural Plasticity

    PubMed Central

    Hampson, Robert E.; Miller, Frances; Palchik, Guillermo; Deadwyler, Sam A.

    2011-01-01

    Chronic activation or inhibition of cannabinoid receptors (CB1) leads to continuous suppression of neuronal plasticity in hippocampus and other brain regions, suggesting that endocannabinoids may have a functional role in synaptic processes that produce state-dependent transient modulation of hippocampal cell activity. In support of this, it has previously been shown in vitro that cannabinoid CB1 receptors modulate second messenger systems in hippocampal neurons that can modulate intracellular ion channels, including channels which release calcium from intracellular stores. Here we demonstrate in hippocampal slices a similar endocannabinoid action on excitatory glutamatergic synapses via modulation of NMDA-receptor mediated intracellular calcium levels in confocal imaged neurons. Calcium entry through glutamatergic NMDA-mediated ion channels increases intracellular calcium concentrations via modulation of release from ryanodine-sensitive channels in endoplasmic reticulum. The studies reported here show that NMDA-elicited increases in Calcium Green fluorescence are enhanced by CB1 receptor antagonists (i.e. rimonabant), and inhibited by CB1 agonists (i.e. WIN 55,212-2). Suppression of endocannabinoid breakdown by either reuptake inhibition (AM404) or fatty-acid amide hydrolase inhibition (URB597) produced suppression of NMDA elicited calcium increases comparable to WIN 55,212-2, while enhancement of calcium release provoked by endocannabinoid receptor antagonists (Rimonabant) was shown to depend on the blockade of CB1 receptor mediated de-phosphorylation of Ryanodine receptors. Such CB1 receptor modulation of NMDA elicited increases in intracellular calcium may account for the respective disruption and enhancement by CB1 agents of trial-specific hippocampal neuron ensemble firing patterns during performance of a short-term memory task, reported previously from this laboratory. PMID:21288475

  9. 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

  10. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    SciTech Connect

    Gean, P.W. )

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  11. The Role of Estrogen Related Receptor in Modulating Estrogen Receptor Mediated Transcription in Breast Cancer Cells

    DTIC Science & Technology

    2005-04-01

    receptors ) by demonstrating that mitochondrial biogenesis and fatty acid P- oxidation , processes ERRa is known to regulate , are robustly...gluconeogenesis, and fatty acid oxidation (Lin 2003; Puigserver 1998; Wu 1999; Yoon 2001). In addition to its activity on a number of nuclear receptors , this...in target cells. They were generated by replacing the receptor interaction domains in peroxisome proliferator activated receptor

  12. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  13. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  14. IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity.

    PubMed

    Zhu, Li X; Sharma, Sherven; Gardner, Brian; Escuadro, Brian; Atianzar, Kimberly; Tashkin, Donald P; Dubinett, Steven M

    2003-04-01

    Sigma receptors are unique endoplasmic reticulum proteins that mediate signaling for a variety of drugs. We determined the effect of sigma(1) receptor agonists on immune responses in a syngeneic lung cancer model. Sigma(1) receptor agonists, including cocaine, up-regulated splenocyte IL-10 mRNA and protein production in vitro in a sigma receptor-dependent, pertussis toxin-sensitive manner. In vivo, sigma(1) receptor agonists promoted tumor growth and induced IL-10 at the tumor site. Increased tumor growth was prevented by administration of specific Abs to IL-10 or by administration of specific sigma(1) receptor antagonists. We report that sigma(1) receptor ligands, including cocaine, augment tumor growth through an IL-10 dependent mechanism.

  15. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

    PubMed Central

    Ling, Ping; Ngo, Karen; Nguyen, Steven; Thurmond, Robin L; Edwards, James P; Karlsson, Lars; Fung-Leung, Wai-Ping

    2004-01-01

    During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H4 but not H3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. Histamine (0.01–30 μM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 μM histamine with EC50 at 19 nM. After 60 min incubation with 1 μM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01–1 μM) also enhanced the eosinophil shape change induced by other chemokines. Histamine-induced eosinophil shape change was mediated by the H4 receptor. This effect was completely inhibited by H4 receptor-specific antagonist JNJ 7777120 (IC50 0.3 μM) and H3/H4 receptor antagonist thioperamide (IC50 1.4 μM), but not by selective H1, H2 or H3 receptor antagonists. H4 receptor agonists imetit (EC50 25 nM) and clobenpropit (EC50 72 nM) could mimic histamine effect in inducing eosinophil shape change. Histamine (0.01–100 μM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 and thioperamide. Histamine (0.01–10 μM) induced eosinophil chemotaxis with an EC50 of 83 nM. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 (IC50 86 nM) and thioperamide (IC50 519 nM). Histamine (0.5 μM) also enhanced the eosinophil shape change induced by other chemokines. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H4 receptor mediates eosinophil chemotaxis, cell shape change and

  16. 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

  17. 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

  18. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    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 apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer 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 apoptotic agents 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 correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  19. 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

  20. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  1. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  2. Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons

    PubMed Central

    Ohtake, Yosuke; Wong, Daniella; Abdul-Muneer, P. M.; Selzer, Michael E.; Li, Shuxin

    2016-01-01

    Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors. PMID:27849007

  3. G protein-coupled receptors: extranuclear mediators for the non-genomic actions of steroids.

    PubMed

    Wang, Chen; Liu, Yi; Cao, Ji-Min

    2014-09-01

    Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs) are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30) for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) for androgen, and trace amine associated receptor 1 (TAAR1) for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid), the structural basis of steroids and GPCRs' interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  4. Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

    PubMed

    Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi

    2015-08-01

    Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

  5. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    SciTech Connect

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  6. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  7. Agonist-mediated assembly of the crustacean methyl farnesoate receptor

    PubMed Central

    Kakaley, Elizabeth K. Medlock; Wang, Helen Y.; LeBlanc, Gerald A.

    2017-01-01

    The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly. PMID:28322350

  8. In silico modelling of prostacyclin and other lipid mediators to nuclear receptors reveal novel thyroid hormone receptor antagonist properties.

    PubMed

    Perez Diaz, Noelia; Zloh, Mire; Patel, Pryank; Mackenzie, Louise S

    2016-01-01

    Prostacyclin (PGI2) is a key mediator involved in cardiovascular homeostasis, acting predominantly on two receptor types; cell surface IP receptor and cytosolic peroxisome proliferator activated receptor (PPAR) β/δ. Having a very short half-life, direct methods to determine its long term effects on cells is difficult, and little is known of its interactions with nuclear receptors. Here we used computational chemistry methods to investigate the potential for PGI2, beraprost (IP receptor agonist), and GW0742 (PPARβ/δ agonist), to bind to nuclear receptors, confirmed with pharmacological methods. In silico screening predicted that PGI2, beraprost, and GW0742 have the potential to bind to different nuclear receptors, in particular thyroid hormone β receptor (TRβ) and thyroid hormone α receptor (TRα). Docking analysis predicts a binding profile to residues thought to have allosteric control on the TR ligand binding site. Luciferase reporter assays confirmed that beraprost and GW0742 display TRβ and TRα antagonistic properties; beraprost IC50 6.3 × 10(-5)mol/L and GW0742 IC50 4.9 × 10(-6) mol/L. Changes to triiodothyronine (T3) induced vasodilation of rat mesenteric arteries measured on the wire myograph were measured in the presence of the TR antagonist MLS000389544 (10(-5) mol/L), beraprost (10(-5) mol/L) and GW0742 (10(-5) mol/L); all significantly inhibited T3 induced vasodilation compared to controls. We have shown that both beraprost and GW0742 exhibit TRβ and TRα antagonist behaviour, and suggests that PGI2 has the ability to affect the long term function of cells through binding to and inactivating thyroid hormone receptors.

  9. 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

  10. The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-d-aspartate receptors

    PubMed Central

    Bacskai, B. J.; Xia, M. Q.; Strickland, D. K.; Rebeck, G. W.; Hyman, B. T.

    2000-01-01

    The low density lipoprotein receptor-related protein (LRP) is an endocytic receptor that is a member of the low density lipoprotein receptor family. We report that the LRP ligand, activated α2-macroglobulin (α2M*), induces robust calcium influx in cultured primary neurons, but not in nonneuronal LRP-containing cells in the same culture. The calcium influx is mediated through N-methyl-d-aspartate receptor channels, which explains the neuron specificity of the response. Microapplication of α2M* leads to a localized response at the site of application that dissipates rapidly, suggesting that the calcium signal is temporally and spatially discrete. Calcium influx to α2M* is blocked by the physiological LRP inhibitor, receptor-associated protein. Bivalent antibodies to the extracellular domain of LRP, but not Fab fragments of the same antibody, cause calcium influx, indicating that the response is specific to LRP and may require dimerization of the receptor. Thus, LRP is an endocytic receptor with a novel signaling role. PMID:11016955

  11. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  12. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

    PubMed

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen

    2009-12-18

    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  13. Androgen receptor serine 81 mediates Pin1 interaction and activity

    PubMed Central

    La Montagna, Raffaele; Caligiuri, Isabella; Maranta, Pasquale; Lucchetti, Chiara; Esposito, Luca; Paggi, Marco G.; Toffoli, Giuseppe; Rizzolio, Flavio; Giordano, Antonio

    2012-01-01

    Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function. PMID:22894932

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

    PubMed

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

    2016-04-29

    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.

  15. 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

  16. Nucleus accumbens μ-opioid receptors mediate social reward.

    PubMed

    Trezza, Viviana; Damsteegt, Ruth; Achterberg, E J Marijke; Vanderschuren, Louk J M J

    2011-04-27

    Positive social interactions are essential for emotional well-being and proper behavioral development of young individuals. Here, we studied the neural underpinnings of social reward by investigating the involvement of opioid neurotransmission in the nucleus accumbens (NAc) in social play behavior, a highly rewarding social interaction in adolescent rats. Intra-NAc infusion of morphine (0.05-0.1 μg) increased pinning and pouncing, characteristic elements of social play behavior in rats, and blockade of NAc opioid receptors with naloxone (0.5 μg) prevented the play-enhancing effects of systemic morphine (1 mg/kg, s.c.) administration. Thus, stimulation of opioid receptors in the NAc was necessary and sufficient for morphine to increase social play. Intra-NAc treatment with the selective μ-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) (0.1-10 ng) and the μ-opioid receptor antagonist Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (0.3-3 μg) increased and decreased social play, respectively. The δ-opioid receptor agonist DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (0.3-3 μg) had no effects, whereas the κ-opioid receptor agonist U69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide) (0.01-1 μg) decreased social play. Intra-NAc treatment with β-endorphin (0.01-1 μg) increased social play, but met-enkephalin (0.1-5 μg) and the enkephalinase inhibitor thiorphan (0.1-1 μg) were ineffective. DAMGO (0.1-10 ng) increased social play after infusion into both the shell and core subregions of the NAc. Last, intra-NAc infusion of CTAP (3 μg) prevented the development of social play-induced conditioned place preference. These findings identify NAc μ-opioid receptor stimulation as an important neural mechanism for the attribution of positive value to social interactions in adolescent rats. Altered NAc μ-opioid receptor function may underlie social impairments in psychiatric disorders such as autism

  17. Excitatory Synaptic Responses Mediated by GABA_A Receptors in the Hippocampus

    NASA Astrophysics Data System (ADS)

    Michelson, Hillary B.; Wong, Robert K. S.

    1991-09-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABA_A receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABA_A receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABA_A receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABA_A receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

  18. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  19. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

    PubMed

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L

    2012-04-01

    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  20. Extranuclear Signaling Effects Mediated by the Estrogen Receptor

    DTIC Science & Technology

    2008-03-01

    inhibition of Pyk2 with salicylate disrupted the ability of E2 to induce the phosphorylation of ERK1/2 without affecting αCaMKII activity (data not...Identification of a phosphorylation site for calcium/calmodulin-dependent protein kinase II in the 13 NR2B subunit of the N- methyl -D-aspartate receptor. J Biol

  1. Ultrasound-Mediated Therapies Using Receptor-Targeted Nanodroplets

    NASA Astrophysics Data System (ADS)

    Schumann, P.; LaBell, R.; Penrose, K.; Kerschen, A.; Unger, E.; Matsunaga, T.; Zutshi, R.

    2006-05-01

    Angiogenic vessels at tumor sites are leaky and allow particles less than 400 nm to pass through. We have developed targeted nanodroplets that are less than 400 nm in size and are capable of encapsulating drug molecules which can be released at the tumor site upon insonation. These nanodroplets are targeted to the α6β1 receptor, which is upregulated in prostate cancer.

  2. Regulation of the androgen receptor by SET9-mediated methylation.

    PubMed

    Gaughan, Luke; Stockley, Jacqueline; Wang, Nan; McCracken, Stuart R C; Treumann, Achim; Armstrong, Kelly; Shaheen, Fadhel; Watt, Kate; McEwan, Iain J; Wang, Chenguang; Pestell, Richard G; Robson, Craig N

    2011-03-01

    The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

  3. 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.

  4. CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells.

    PubMed

    Zhou, D; Song, Z H

    2001-08-15

    The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain.

  5. Crosstalk between Leptin Receptor and Igf-Ir in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2008-04-01

    AD_________________ Award Number: W81XWH-06-1-0452 TITLE: Crosstalk between Leptin Receptor and Igf...NUMBER Crosstalk between Leptin Receptor and Igf-Ir in Breast Cancer: A Potential Mediator of Chemoresistance 5b. GRANT NUMBER W81XWH-06-1...cancer, and is associated with reduced treatment response and reduced overall survival. The obesity-associated hormones IGF-I and leptin and their

  6. Crosstalk Between Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2009-04-01

    5). Leptin , a product of the obese (ob) gene, is an adipocytokine that regulates appetite , bone formation, reproduction, cellular proliferation, and...AD_________________ Award Number: W81XWH-06-1-0452 TITLE: Crosstalk Between Leptin Receptor and... Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance 5b. GRANT NUMBER W81XWH-06-1-0452 5c. PROGRAM ELEMENT NUMBER

  7. 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.

  8. 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.

  9. Orchestration of salivary secretion mediated by two different dopamine receptors in the blacklegged tick Ixodes scapularis

    PubMed Central

    Kim, Donghun; Šimo, Ladislav; Park, Yoonseong

    2014-01-01

    Salivary secretion is crucial for successful tick feeding, and it is the mediator of pathogen transmission. Salivation functions to inhibit various components of the host immune system and remove excess water and ions during the ingestion of large blood meals. Control of salivary glands involves autocrine/paracrine dopamine, which is the most potent inducer of tick salivation. Previously, we reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Here, we investigated the different physiological roles of the dopamine receptors in tick salivary glands by using pharmacological tools that discriminate between the two distinct receptors. Heterologous expressions followed by reporter assays of the dopamine receptors identified receptor-specific antagonists and agonists. These pharmacological tools were further used to discriminate the physiological role of each receptor by using in vitro assays: measuring salivary secretions of isolated salivary glands and monitoring dynamic changes in the size of individual salivary gland acini. We propose that the D1 receptor acts on salivary gland acini epithelial cells for inward fluid transport. InvD1L controls (or modulates) each acinus for expelling saliva from the acini to the salivary ducts, presumably through the actions of myoepithelial cells and valves for pumping/gating. We conclude that dopamine acts on the D1 and the InvD1L receptors and leads different physiological actions to orchestrate tick salivary secretion. PMID:25320269

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

    PubMed

    Mattila, Pieta K; Batista, Facundo D; Treanor, Bebhinn

    2016-02-01

    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.

  11. Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

    PubMed

    Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

    2014-03-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters.

  12. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  13. P2 receptor-mediated signaling in mast cell biology.

    PubMed

    Bulanova, Elena; Bulfone-Paus, Silvia

    2010-03-01

    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  14. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    PubMed Central

    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. PMID:25901833

  15. Visualizing clathrin-mediated IgE receptor internalization by electron and atomic force microscopy.

    PubMed

    Burns, Alan R; Oliver, Janet M; Pfeiffer, Janet R; Wilson, Bridget S

    2008-01-01

    A significant step in the immunoglobulin E (IgE) receptor signaling pathway in mast cell membranes is receptor internalization by clathrin-coated vesicles. Visualization in native membrane sheets of the emerging clathrin lattice structures containing the IgE receptor and associated signaling partners has been accomplished with high-resolution transmission electron microscopy (TEM). More recently, membrane sheets with labeled clathrin have also been characterized with atomic force microscopy (AFM) in combination with fluorescence imaging. We discuss here the procedure for creating fixed, native cell membrane sheets, labeling with immunogold or fluorescent labels, and utilization for TEM or AFM/fluorescence imaging of clathrin-mediated IgE internalization.

  16. An Fcγ receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells.

    PubMed

    Wilson, Nicholas S; Yang, Becky; Yang, Annie; Loeser, Stefanie; Marsters, Scot; Lawrence, David; Li, Yun; Pitti, Robert; Totpal, Klara; Yee, Sharon; Ross, Sarajane; Vernes, Jean-Michel; Lu, Yanmei; Adams, Cam; Offringa, Rienk; Kelley, Bob; Hymowitz, Sarah; Daniel, Dylan; Meng, Gloria; Ashkenazi, Avi

    2011-01-18

    Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

  17. P2X receptors as targets for the treatment of status epilepticus

    PubMed Central

    Henshall, David C.; Diaz-Hernandez, Miguel; Miras-Portugal, M. Teresa; Engel, Tobias

    2013-01-01

    Prolonged seizures are amongst the most common neurological emergencies. Status epilepticus is a state of continuous seizures that is life-threatening and prompt termination of status epilepticus is critical to protect the brain from permanent damage. Frontline treatment comprises parenteral administration of anticonvulsants such as lorazepam that facilitate γ-amino butyric acid (GABA) transmission. Because status epilepticus can become refractory to anticonvulsants in a significant proportion of patients, drugs which act on different neurotransmitter systems may represent potential adjunctive treatments. P2X receptors are a class of ligand-gated ion channel activated by ATP that contributes to neuro- and glio-transmission. P2X receptors are expressed by both neurons and glia in various brain regions, including the hippocampus. Electrophysiology, pharmacology and genetic studies suggest certain P2X receptors are activated during pathologic brain activity. Expression of several members of the family including P2X2, P2X4, and P2X7 receptors has been reported to be altered in the hippocampus following status epilepticus. Recent studies have shown that ligands of the P2X7 receptor can have potent effects on seizure severity during status epilepticus and mice lacking this receptor display altered seizures in response to chemoconvulsants. Antagonists of the P2X7 receptor also modulate neuronal death, microglial responses and neuroinflammatory signaling. Recent work also found altered neuronal injury and inflammation after status epilepticus in mice lacking the P2X4 receptor. In summary, members of the P2X receptor family may serve important roles in the pathophysiology of status epilepticus and represent novel targets for seizure control and neuroprotection. PMID:24324404

  18. An histidine covalent receptor/butenolide complex mediates strigolactone perception

    PubMed Central

    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

    2016-01-01

    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 characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand. PMID:27479744

  19. Taste Receptors Mediate Sinonasal Immunity and Respiratory Disease

    PubMed Central

    Douglas, Jennifer E.; Cohen, Noam A.

    2017-01-01

    The bitter taste receptor T2R38 has been shown to play a role in the pathogenesis of chronic rhinosinusitis (CRS), where the receptor functions to enhance upper respiratory innate immunity through a triad of beneficial immune responses. Individuals with a functional version of T2R38 are tasters for the bitter compound phenylthiocarbamide (PTC) and exhibit an anti-microbial response in the upper airway to certain invading pathogens, while those individuals with a non-functional version of the receptor are PTC non-tasters and lack this beneficial response. The clinical ramifications are significant, with the non-taster genotype being an independent risk factor for CRS requiring surgery, poor quality-of-life (QOL) improvements post-operatively, and decreased rhinologic QOL in patients with cystic fibrosis. Furthermore, indirect evidence suggests that non-tasters also have a larger burden of biofilm formation. This new data may influence the clinical management of patients with infectious conditions affecting the upper respiratory tract and possibly at other mucosal sites throughout the body. PMID:28218655

  20. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-03-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago).

  1. Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

    PubMed

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

    2011-02-11

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

  2. 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

  3. Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

    PubMed Central

    Kolodkin, Alexey; Sahin, Nilgun; Phillips, Anna; Hood, Steve R.; Bruggeman, Frank J.; Westerhoff, Hans V.; Plant, Nick

    2013-01-01

    It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress. PMID:23653204

  4. Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

    DTIC Science & Technology

    2012-10-01

    brain edema. Methods Mice. Aqp4−/− mice were generated by GenOway by cloning and sequencing of a targeted region of the murine Aqp4 gene in a 129/Sv... genetic back- ground. The strategy was to design a targeted locus allowing us to delete exons 1–3 to avoid any expression of putative splice variants...was made on the back region and a laminectomy was performed aseptically at the T11-T12 level. Before SCI, a catheter was placed in the left femoral

  5. Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

    DTIC Science & Technology

    2012-10-01

    myelination ef- ficiency, sheath thickness , and G-ratios during development. These observations suggest that PTPRZ1 inhibition might comprise a...The animals were sacrificed at 24 hrs, and SC tissue was dissociated with papain. A Percoll gradient was performed to remove myelin , debris and...year 2 (2011-12) Goldman, S.A. Progenitor cell-based treatment of the pediatric myelin disorders. Arch. Neurology, 68:848-56, 2011. Bugiani, M

  6. 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

  7. Neuroprotection Mediated by P2Y13 Nucleotide Receptors in Neurons

    PubMed Central

    Pérez-Sen, Raquel; Queipo, Mª José; Morente, Verónica; Ortega, Felipe; Delicado, Esmerilda G.; Miras-Portugal, Mª Teresa

    2015-01-01

    ADP-specific P2Y13 receptor constitutes one of the most recently identified nucleotide receptor and the understanding of their physiological role is currently under investigation. Cerebellar astrocytes and granule neurons provide excellent models to study P2Y13 expression and function since the first identification of ADP-evoked calcium responses not attributable to the related P2Y1 receptor was performed in these cell populations. In this regard, all responses induced by ADP analogues in astrocytes resulted to be Gi-coupled activities mediated by P2Y13 instead of P2Y1 receptors. Similarly, both glycogen synthase kinase-3 (GSK3) and ERK1/2 signaling triggered by 2MeSADP in cerebellar granule neurons were also dependent on Gi-coupled receptors, and mediated by PI3K activity. In granule neurons, P2Y13 receptor was specifically coupled to the main neuronal survival PI3K/Akt-cascade targeting GSK3 phosphorylation. GSK3 inhibition led to nuclear translocation of transcriptional targets, including β-catenin and Nrf2. The activation of the Nrf2/heme oxygenase-1 (HO-1) axis was responsible for the prosurvival effect against oxidative stress. In addition, P2Y13-mediated ERK1/2 signaling in granule neurons also triggered activation of transcription factors, such as CREB, which underlined the antiapoptotic action against glutamate-induced excitotoxicity. Finally, a novel signaling mechanism has been recently described for a P2Y13 receptor in granule neurons that involved the expression of a dual protein phosphatase, DUSP2. This activity contributed to regulate MAPK activation after genotoxic stress. In conclusion, P2Y13 receptors harbored in cerebellar astrocytes and granule neurons exhibit specific signaling properties that link them to specialized functions at the level of neuroprotection and trophic activity in both cerebellar cell populations. PMID:25750704

  8. Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

    PubMed Central

    Knutson, Todd P.; Lange, Carol A.

    2014-01-01

    Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR’s contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered along side antiestrogens and aromatase inhibitors. PMID:24291072

  9. 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.

  10. Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

    PubMed

    Andjelic, S; Liou, H C

    1998-02-01

    An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

  11. Lysosphingolipid receptor-mediated diuresis and natriuresis in anaesthetized rats.

    PubMed

    Bischoff, A; Meyer Zu Heringdorf, D; Jakobs, K H; Michel, M C

    2001-04-01

    Lysosphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act on specific G-protein-coupled receptors. Since SPP and SPPC cause renal vasoconstriction, we have investigated their effects on urine and electrolyte excretion in anaesthetized rats. Infusion of SPP (1 - 30 microg kg(-1) min(-1)) for up to 120 min dose-dependently but transiently (peak after 15 min, disappearance after 60 min) reduced renal blood flow without altering endogenous creatinine clearance. Nevertheless, infusion of SPP increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. These tubular lysosphingolipid effects developed more slowly (maximum after 60 - 90 min) and also abated more slowly upon lysosphingolipid washout than the renovascular effects. Infusion of SPPC, sphingosine and glucopsychosine (3 - 30 microg kg(-1) min(-1) each) caused little if any alterations in renal blood flow but also increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. Pretreatment with pertussis toxin (10 microg kg(-1) 3 days before the acute experiment) abolished the renovascular and tubular effects of 30 microg kg(-1) min(-1) SPP. These findings suggest that lysosphingolipids are a hitherto unrecognized class of endogenous modulators of renal function. SPP affects renovascular tone and tubular function via receptors coupled to G(i)-type G-proteins. SPPC, sphingosine and glucopsychosine mimic only the tubular effects of SPP, and hence may act on distinct sites.

  12. Toll-Like Receptor-9-Mediated Invasion in Breast Cancer

    DTIC Science & Technology

    2011-07-01

    and purify TLR9 in-house, recombinant hTLR9 is now available for purchase from BioClone, Inc., San Diego, CA (http://www.bioclone.us/CD- Marker -cDNA...structure dominates at lower pH, even in the presence of salt. Correlation of DNA structure and stabilit y with TLR9-mediated cancer cell in vasion...TTAGGG)3], was examined. This sequence is found at the ends of human chromosomes and due to the nature of the repetitive GGGs forms G-tetrads that

  13. 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

  14. Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

    PubMed Central

    Daray, Federico Manuel; Minvielle, Ana Itatí; Puppo, Soledad; Rothlin, Rodolfo Pedro

    2003-01-01

    This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration–response curves to U-46619 (TXA2 mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC50: 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pKB values of 7.96 and 9.07, respectively.Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17-phenyl-trinor-PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17-phenyl-trinor-PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI-192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively.Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed.In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel. PMID:12922927

  15. 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

  16. Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses.

    PubMed

    Hayashi, Chie; Madrigal, Andres G; Liu, Xinyan; Ukai, Takashi; Goswami, Sulip; Gudino, Cynthia V; Gibson, Frank C; Genco, Caroline A

    2010-01-01

    Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotein E-deficient (ApoE(-/-)) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-gamma and the pro-inflammatory cytokines IL-1 beta, IL-6 and tumor necrosis factor-alpha in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2(-/-)) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen.

  17. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  18. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression

    PubMed Central

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-IL; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y.L.; Choi, Hueng-Sik

    2017-01-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ -binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism. PMID:26348907

  19. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression.

    PubMed

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-Il; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y L; Choi, Hueng-Sik

    2015-09-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism.

  20. 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

  1. 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

  2. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles

    PubMed Central

    Hu, Xiaoming; Liou, Anthony K.F.; Leak, Rehana K.; Xu, Mingyue; An, Chengrui; Suenaga, Jun; Shi, Yejie; Gao, Yanqin; Zheng, Ping; Chen, Jun

    2014-01-01

    Microglia are the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery and are known for their ability to assume diverse phenotypes. A broad range of surface receptors are expressed on microglia and mediate microglial ‘On’ or ‘Off’ responses to signals from other host cells as well as invading microorganisms. The integrated actions of these receptors result in tightly regulated biological functions, including cell mobility, phagocytosis, the induction of acquired immunity, and trophic factor/inflammatory mediator release. Over the last few years, significant advances have been made towards deciphering the signaling mechanisms related to these receptors and their specific cellular functions. In this review, we describe the current state of knowledge of the surface receptors involved in microglial activation, with an emphasis on their engagement of distinct functional programs and their roles in CNS injuries. It will become evident from this review that microglial homeostasis is carefully maintained by multiple counterbalanced strategies, including, but not limited to, ‘On’ and ‘Off’ receptor signaling. Specific regulation of theses microglial receptors may be a promising therapeutic strategy against CNS injuries. PMID:24923657

  3. A slow excitatory postsynaptic current mediated by a novel metabotropic glutamate receptor in CA1 pyramidal neurons.

    PubMed

    Sheng, Nengyin; Yang, Jing; Silm, Katlin; Edwards, Robert H; Nicoll, Roger A

    2017-03-15

    Slow excitatory postsynaptic currents (EPSCs) mediated by metabotropic glutamate receptors (mGlu receptors) have been reported in several neuronal subtypes, but their presence in hippocampal pyramidal neurons remains elusive. Here we find that in CA1 pyramidal neurons a slow EPSC is induced by repetitive stimulation while ionotropic glutamate receptors and glutamate-uptake are blocked whereas it is absent in the VGLUT1 knockout mouse in which presynaptic glutamate is lost, suggesting the slow EPSC is mediated by glutamate activating mGlu receptors. However, it is not inhibited by known mGlu receptor antagonists. These findings suggest that this slow EPSC is mediated by a novel mGlu receptor, and that it may be involved in neurological diseases associated with abnormal high-concentration of extracellular glutamate. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

  4. 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).

  5. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    PubMed

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  6. 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

  7. 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.

  8. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

    PubMed Central

    Fan, Xueliang; Hess, Ellen J.

    2007-01-01

    The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. PMID:17291774

  9. Oseltamivir blocks human neuronal nicotinic acetylcholine receptor-mediated currents.

    PubMed

    Muraki, Katsuhiko; Hatano, Noriyuki; Suzuki, Hiroka; Muraki, Yukiko; Iwajima, Yui; Maeda, Yasuhiro; Ono, Hideki

    2015-02-01

    The effects of oseltamivir, a neuraminidase inhibitor, were tested on the function of neuronal nicotinic acetylcholine receptors (nAChRs) in a neuroblastoma cell line IMR32 derived from human peripheral neurons and on recombinant human α3β4 nAChRs expressed in HEK cells. IMR32 cells predominately express α3β4 nAChRs. Nicotine (nic, 30 μm)-evoked currents recorded at -90 mV in IMR32 cells using the whole-cell patch clamp technique were reversibly blocked by oseltamivir in a concentration-dependent manner. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) at 30 μm had little effect on the nic-evoked currents. Oseltamivir also blocked nic-evoked currents derived from HEK cells with recombinant α3β4 nAChRs. This blockade was voltage-dependent with 10, 30 and 100 μm oseltamivir inhibiting ~50% at -100, -60 and -40 mV, respectively. Non-inactivating currents in IMR32 cells and in HEK cells with α3β4 nAChRs, which were evoked by an endogenous nicotinic agonist, ACh (5 μm), were reversibly blocked by oseltamivir. These data demonstrate that oseltamivir blocks nAChRs, presumably via binding to a site in the channel pore.

  10. The mannose receptor mediates dengue virus infection of macrophages.

    PubMed

    Miller, Joanna L; de Wet, Barend J M; deWet, Barend J M; Martinez-Pomares, Luisa; Radcliffe, Catherine M; Dwek, Raymond A; Rudd, Pauline M; Gordon, Siamon

    2008-02-08

    Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

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

    PubMed

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

    2015-07-10

    Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating.

  13. Ryanodine receptor-mediated arrhythmias and sudden cardiac death