Behavioral effects of gamma-hydroxybutyrate, its precursor gamma-butyrolactone, and GABA(B) receptor agonists: time course and differential antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348).

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Koek, Wouter; Mercer, Susan L; Coop, Andrew; France, Charles P

2009-09-01

Gamma-hydroxybutyrate (GHB) is used therapeutically and recreationally. The mechanism by which GHB produces its therapeutic and recreational effects is not entirely clear, although GABA(B) receptors seem to play an important role. This role could be complex, because there are indications that different GABA(B) receptor mechanisms mediate the effects of GHB and the prototypical GABA(B) receptor agonist baclofen. To further explore possible differences in underlying GABA(B) receptor mechanisms, the present study examined the effects of GHB and baclofen on operant responding and their antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348). Pigeons were trained to peck a key for access to food during response periods that started at different times after the beginning of the session. In these pigeons, GHB, its precursor gamma-butyrolactone (GBL), and the GABA(B) receptor agonists baclofen and 3-aminopropyl(methyl)phosphinic acid hydrochloride (SKF97541) decreased the rate of responding in a dose- and time-dependent manner. CGP35348 shifted the dose-response curve of each agonist to the right, but the magnitude of the shift differed among the agonists. Schild analysis yielded a pA(2) value of CGP35348 to antagonize GHB and GBL [i.e., 3.9 (3.7-4.2)] that was different (P = 0.0011) from the pA(2) value to antagonize baclofen and SKF97541 [i.e., 4.5 (4.4-4.7)]. This finding is further evidence that the GABA(B) receptor mechanisms mediating the effects of GHB and prototypical GABA(B) receptor agonists are not identical. A better understanding of the similarities and differences between these mechanisms, and their involvement in the therapeutic effects of GHB and baclofen, could lead to more effective medications with fewer adverse effects.

Low 5-HT1B receptor binding in the migraine brain

DEFF Research Database (Denmark)

Deen, Marie; Hansen, Hanne D; Hougaard, Anders

2018-01-01

Background The pathophysiology of migraine may involve dysfunction of serotonergic signaling. In particular, the 5-HT1B receptor is considered a key player due to the efficacy of 5-HT1B receptor agonists for treatment of migraine attacks. Aim To examine the cerebral 5-HT1B receptor binding....... Patients who reported migraine brain regions involved in pain modulation as regions of interest and applied a latent variable model (LVM) to assess the group effect on binding across these regions. Results Our data...... support a model wherein group status predicts the latent variable ( p = 0.038), with migraine patients having lower 5-HT1B receptor binding across regions compared to controls. Further, in a whole-brain voxel-based analysis, time since last migraine attack correlated positively with 5-HT1B receptor...

The Role of PPAR Receptors and Leukotriene B4 Receptors in Mediating the Effects of LY293111 in Pancreatic Cancer

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Thomas E. Adrian

2008-01-01

Full Text Available Pancreatic cancer is a devastating disease in which current therapies are inadequate. Separate lines of research have identified the 5-lipoxygenase/leukotriene B4 receptor pathway and the PPAR pathway as potential targets for prevention or treatment of this disease. LY293111 was originally designed as a potent leukotriene B4 receptor antagonist for treatment of inflammatory conditions. LY293111 was also known to have inhibitory effects on 5-lipoxygenase, which is upstream of the production of leukotrienes. LY293111 was shown to have potent anticancer effects in pancreatic cancer and several other solid malignancies, where it caused cell cycle arrest and marked apoptosis. Subsequently, it came to light that LY293111 exhibited PPAR agonist activity in addition to its effects on the 5-lipoxygenase pathway. This raises the question of which of the two targets is of greatest importance with regard to the anticancer effects of this agent. The evidence to date is not conclusive, but suggests that the effects of LY293111 may be mediated by both LTB4 receptors and PPAR.

Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2.

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Sebastien A Gauthier

Full Text Available The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR and Ultraspiracle (USP. In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4 is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC, the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH, an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

International Nuclear Information System (INIS)

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.; Mantey, S.A.; Gardner, J.D.; Jensen, R.T.

1989-01-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-[Tyr4]bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-[Tyr4]bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-[Tyr4]bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were as follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-[Tyr4]bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B

Differential involvement of 5-HT(1A) and 5-HT(1B/1D) receptors in human interferon-alpha-induced immobility in the mouse forced swimming test.

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Zhang, Hongmei; Wang, Wei; Jiang, Zhenzhou; Shang, Jing; Zhang, Luyong

2010-01-01

Although Interferon-alpha (IFN-alpha, CAS 9008-11-1) is a powerful drug in treating several viral infections and certain tumors, a considerable amount of neuropsychiatric side-effects such as depression and anxiety are an unavoidable consequence. Combination with the selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine (CAS 56296-78-7) significantly improved the situation. However, the potential 5-HT(1A) receptor- and 5-HT(1B) receptor-signals involved in the antidepressant effects are still unclear. The effects of 5-HT(1A) receptor- and 5-HT(1B) receptor signals were analyzed by using the mouse forced swimming test (FST), a predictive test of antidepressant-like action. The present results indicated that (1) fluoxetine (administrated intragastrically, 30 mg/kg; not subactive dose: 15 mg/kg) significantly reduced IFN-alpha-induced increase of the immobility time in the forced swimming test; (2) 5-HT(1A) receptor- and 5-HT(1B) receptor ligands alone or in combination had no effects on IFN-alpha-induced increase of the immobility time in the FST; (3) surprisingly, WAY 100635 (5-HT(1A) receptor antagonist, 634908-75-1) and 8-OH-DPAT(5-HT(1A) receptor agonist, CAS 78950-78-4) markedly enhanced the antidepressant effect of fluoxetine at the subactive dose (15 mg/kg, i. g.) on the IFN-alpha-treated mice in the FST. Further investigations showed that fluoxetine combined with WAY 100635 and 8-OH-DPAT failed to produce antidepressant effects in the FST. (4) Co-application of CGS 12066A (5-HT(1B) receptor agonist, CAS 109028-09-3) or GR 127935 (5-HT(1B/1D) receptor antagonist, CAS 148642-42-6) with fluoxetine had no synergistic effects on the IFN-alpha-induced increase of immobility time in FST. (5) Interestingly, co-administration of GR 127935, WAY 100635 and fluoxetine significantly reduced the IFN-alpha-induced increase in immobility time of FST, being more effective than co-administration of WAY 100635 and fluoxetine. All results suggest that (1) compared to

NK cell cytotoxicity mediated by 2B4 and NTB-A is dependent on SAP acting downstream of receptor phosphorylation

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Stephan eMeinke

2013-01-01

Full Text Available 2B4 (CD244 and NK-T-B-antigen (NTB-A, CD352 are activating receptors on human NK cells and belong to the family of SLAM-related receptors. Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SAP and EAT-2. X-linked lymphoproliferative syndrome (XLP is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.

Role of kinin B1 and B2 receptors in memory consolidation during the aging process of mice.

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Lemos, Mayra Tolentino Resk; Amaral, Fabio Agostini; Dong, Karis Ester; Bittencourt, Maria Fernanda Queiroz Prado; Caetano, Ariadiny Lima; Pesquero, João Bosco; Viel, Tania Araujo; Buck, Hudson Sousa

2010-04-01

Under physiological conditions, elderly people present memory deficit associated with neuronal loss. This pattern is also associated with Alzheimer's disease but, in this case, in a dramatically intensified level. Kinin receptors have been involved in neurodegeneration and increase of amyloid-beta concentration, associated with Alzheimer's disease (AD). Considering these findings, this work evaluated the role of kinin receptors in memory consolidation during the aging process. Male C57Bl/6 (wt), knock-out B1 (koB1) or B2 (koB2) mice (3, 6, 12 and 18-month-old - mo; n=10 per group) were submitted to an acquisition session, reinforcement to learning (24h later: test 1) and final test (7days later: test 2), in an active avoidance apparatus, to evaluate memory. Conditioned avoidance responses (CAR, % of 50 trials) were registered. In acquisition sessions, similar CAR were obtained among age matched animals from all strains. However, a significant decrease in CAR was observed throughout the aging process (3mo: 8.8+/-2.3%; 6mo: 4.1+/-0.6%; 12mo: 2.2+/-0.6%, 18mo: 3.6+/-0.6%, Pprocess. In test 1, as expected, memory retention increased significantly (Pmemory retention. In test 2, 3- and 6-month-old wt and koB1 mice of all ages showed a significant improvement in memory (Pmemory retention. We suggest that, during the aging process, the B1 receptor could be involved in neurodegeneration and memory loss. Nevertheless, the B2 receptor is apparently acting as a neuroprotective factor. Copyright 2009 Elsevier Ltd. All rights reserved.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

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Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M 1 -, M 2 - and M 4 -subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo . Our previous results show that M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

International Nuclear Information System (INIS)

Lee, Yen-Ching; Perren, Janeanne R; Douglas, Evelyn L; Raynor, Michael P; Bartley, Maria A; Bardy, Peter G; Stephenson, Sally-Anne

2005-01-01

The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target

Induction of the nuclear IκB protein IκB-ζ upon stimulation of B cell antigen receptor

International Nuclear Information System (INIS)

Hijioka, Kuniaki; Matsuo, Susumu; Eto-Kimura, Akiko; Takeshige, Koichiro; Muta, Tatsushi

2007-01-01

The nuclear IκB protein IκB-ζ is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced IκB-ζ associates with nuclear factor (NF)-κB in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of IκB-ζ in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, IκB-ζ mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-κB and induced its target gene, IκB-α, co-crosslinking of Fcγ receptor IIB to the surface immunoglobulin complex inhibited NF-κB activation and the induction of IκB-ζ and IκB-α, suggesting critical roles for NF-κB in the induction. These results indicate that IκB-ζ is also induced by stimulation of B cell antigen receptor, suggesting that IκB-ζ is involved in the regulation of adaptive immune responses

NR4A nuclear receptors are orphans but not lonesome.

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Kurakula, Kondababu; Koenis, Duco S; van Tiel, Claudia M; de Vries, Carlie J M

2014-11-01

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein-protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77. Copyright © 2014 Elsevier B.V. All rights reserved.

Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses.

Science.gov (United States)

Gronert, K; Martinsson-Niskanen, T; Ravasi, S; Chiang, N; Serhan, C N

2001-01-01

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

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Margaret Veselits

Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

NF-kappaB signaling mediates vascular smooth muscle endothelin type B receptor expression in resistance arteries

DEFF Research Database (Denmark)

Zheng, Jian-Pu; Zhang, Yaping; Edvinsson, Lars

2010-01-01

Vascular smooth muscle cells (SMC) endothelin type B (ET(B)) receptor upregulation results in strong vasoconstriction and reduction of local blood flow. We hypothesizes that the underlying molecular mechanisms involve transcriptional factor nuclear factor-kappaB (NF-kappaB) pathway. ET(B) recepto...

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

International Nuclear Information System (INIS)

Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

2010-01-01

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 μM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 μM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 μM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 μM and 10 μM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

Science.gov (United States)

Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

2010-06-15

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.

Ectodomains of the LDL receptor-related proteins LRP1b and LRP4 have anchorage independent functions in vivo.

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Martin F Dietrich

2010-04-01

Full Text Available The low-density lipoprotein (LDL receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL receptor-related protein 1b (LRP1B was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD is fully viable with no apparent phenotype.Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and gamma-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain.Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b or perinatally (Lrp4 lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby

Toll-like receptor 7 cooperates with IL-4 in activated B cells through antigen receptor or CD38 and induces class switch recombination and IgG1 production.

Science.gov (United States)

Tsukamoto, Yumiko; Nagai, Yoshinori; Kariyone, Ai; Shibata, Takuma; Kaisho, Tsuneyasu; Akira, Shizuo; Miyake, Kensuke; Takatsu, Kiyoshi

2009-04-01

IL-4 and 8-mercaptoguanosine (8-SGuo) stimulation of CD38-activated B cells induces mu to gamma1 class switch recombination (CSR) at the DNA level leading to a high level of IgG1 production. Although some of signaling events initiated by IL-4 in activated B cells have been characterized, the involvement of TLR/MyD88 and Btk pathway in IL-4-dependent mu to gamma1 CSR has not been thoroughly evaluated. In this study, we characterized receptors for 8-SGuo and differential roles of 8-SGuo and IL-4 in the induction and mu to gamma1 CSR and IgG1 production. The role of TLR7 and MyD88 in 8-SGuo-induced AID expression and mu to gamma1 CSR was documented, as 8-SGuo did not act on CD38-stimulated splenic B cells from Tlr7(-/-) and Myd88(-/-) mice. CD38-activated B cells from Btk-deficient mice failed to respond to TLR7 ligands for the AID expression and CSR, indicating that Btk is also indispensable for the system. Stimulation of CD38-activated B cells with 8-SGuo induced significant AID expression and DNA double strand breaks, but IL-4 stimulation by itself did not trigger mu to gamma1 CSR. Intriguingly, the mu to gamma1 CSR in the B cells stimulated with CD38 and 8-SGuo totally depends on IL-4 stimulation. Similar results were obtained in the activated B cells through BCR and loxoribine, a well-known TLR7 ligand, in place of 8-SGuo. In vivo administration of TLR7 ligand and anti-CD38 antibody induced the generation of CD138(+) IgG1(+) cells. These results indicate that TLR7 is a receptor for 8-SGuo and plays an essential role in the AID and Blimp-1 expression; however it is not enough to complete mu to gamma1 CSR in CD38-activated B cells. IL-4 may be required for the induction of DNA repair system together with AID for the completion of CSR.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Science.gov (United States)

Napier, Susan E; Letourneau, Jeffrey J; Ansari, Nasrin; Auld, Douglas S; Baker, James; Best, Stuart; Campbell-Wan, Leigh; Chan, Ray; Craighead, Mark; Desai, Hema; Ho, Koc-Kan; MacSweeney, Cliona; Milne, Rachel; Richard Morphy, J; Neagu, Irina; Ohlmeyer, Michael H J; Pick, Jack; Presland, Jeremy; Riviello, Chris; Zanetakos, Heather A; Zhao, Jiuqiao; Webb, Maria L

2011-06-15

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction. Copyright © 2011 Elsevier Ltd. All rights reserved.

B cell receptor signaling pathway involved in benign lymphoepithelial lesions of the lacrimal gland

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Xiao-Na Wang

2017-05-01

Full Text Available AIM: To detect the expression of B cell receptor signaling pathway (BCRSP in lacrimal gland benign lymphoepithelial lesions (LGBLEL. METHODS: Gene microarray was used to compare whole-genome expression in lacrimal gland tissues from LGBLEL patients to tissues from orbital cavernous hemangioma (control tissues. Expression of BCRSP was confirmed by polymerase chain reaction (PCR and immunohistochemistry. RESULTS: The expression of 22 genes of the BCRSP increased significantly in LGBLEL patients. PCR analysis showed that CD22, CR2, and BTK were all highly expressed in LGBLEL tissues. Immunohistochemical analysis showed that CR2 protein was present in LGBLEL, but CD22 and BTK proteins were negative. CR2, CD22, and BTK were not observed in the orbital cavernous hemangiomas with either PCR or immunohistochemistry. CONCLUSION: BCRSP might be involved in the pathogenesis of LGBLEL.

Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

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Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

2016-06-23

The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors

Serotonin 2B Receptors in Mesoaccumbens Dopamine Pathway Regulate Cocaine Responses.

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Doly, Stéphane; Quentin, Emily; Eddine, Raphaël; Tolu, Stefania; Fernandez, Sebastian P; Bertran-Gonzalez, Jesus; Valjent, Emmanuel; Belmer, Arnauld; Viñals, Xavier; Callebert, Jacques; Faure, Philippe; Meye, Frank J; Hervé, Denis; Robledo, Patricia; Mameli, Manuel; Launay, Jean-Marie; Maldonado, Rafael; Maroteaux, Luc

2017-10-25

Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT 2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT 2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT 2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT 2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT 2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT 2B -receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT 2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse. SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT 2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT 2B receptors in a subpopulation of

Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

International Nuclear Information System (INIS)

Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake; Ozaki, Norio; Noda, Yukihiro

2016-01-01

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100 μM) and doxorubicin (0.2 µM) decreased the cell survival rate, but olanzapine (1–100 µM) did not. Under granulocytic differentiation for 5 days, clozapine, even at a concentration of 25 μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H 4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H 4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H 4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H 4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation. - Highlights: • HL-60

TLR4/MyD88/NF-κB signaling and PPAR-γ within the paraventricular nucleus are involved in the effects of telmisartan in hypertension

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Li, Hong-Bao; Li, Xiang; Huo, Chan-Juan; Su, Qing; Guo, Jing [Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an Jiaotong University Cardiovascular Research Center, Xi' an 710061 (China); Yuan, Zu-Yi [Department of Cardiovascular Medicine, First Affiliated Hospital of Medical College of Xi' an Jiaotong University, Xi' an 710061 (China); Zhu, Guo-Qing [Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029 (China); Shi, Xiao-Lian, E-mail: shxl@mail.xjtu.edu.cn [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an 710061 (China); Liu, Jin-Jun, E-mail: jupet@163.com [Department of Physiology and Pathophysiology, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an 710061 (China); Kang, Yu-Ming, E-mail: ykang@mail.xjtu.edu.cn [Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an Jiaotong University Cardiovascular Research Center, Xi' an 710061 (China)

2016-08-15

Previous findings from our laboratory and others indicate that the main therapeutic effect of angiotensin II type 1 receptor (AT1-R) antagonists is to decrease blood pressure and exert anti-inflammatory effects in the cardiovascular system. In this study, we determined whether AT1-R antagonist telmisartan within the hypothalamic paraventricular nucleus (PVN) attenuates hypertension and hypothalamic inflammation via both the TLR4/MyD88/NF-κB signaling pathway and peroxisome proliferator-activated receptor-γ (PPAR-γ) in the PVN in hypertensive rats. Spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were treated for 4 weeks through bilateral PVN infusion with the AT1-R antagonist telmisartan (TEL, 10 μg/h), or losartan (LOS, 20 μg/h), or the PPAR-γ antagonist GW9662 (GW, 100 μg/h), or vehicle via osmotic minipump. Mean arterial pressure (MAP) was recorded by a tail-cuff occlusion method. PVN tissue and blood were collected for the measurement of AT1-R, PPAR-γ, pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6), inducible nitric oxide synthase (iNOS), TLR4, MyD88, nuclear factor-kappa B (NF-κB) activity and plasma norepinephrine (NE), respectively. Hypertensive rats exhibited significantly higher level of AT1-R and lower level of PPAR-γ in the PVN. PVN treatment with TEL attenuated MAP, improved cardiac hypertrophy, reduced TNF-α, IL-1β, IL-6, iNOS levels, and plasma NE in SHR but not in WKY rats. These results were associated with reduced TLR4, MyD88 and NF-κB levels and increased PPAR-γ level in the PVN of hypertensive rats. Our findings suggest that TLR4/MyD88/NF-κB signaling and PPAR-γ within the PVN are involved in the beneficial effects of telmisartan in hypertension. - Highlights: • PVN infusion of TEL in spontaneously hypertensive rats is reported. • PVN infusion of TEL attenuates hypertension and proinflammatory cytokines in PVN. • PVN blockade of AT1-R attenuates

Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

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Yoon, SeongHun; Gariepy, Cheryl E; Yanagisawa, Masashi; Zuccarello, Mario; Rapoport, Robert M

2016-03-01

The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

LPS-Toll-Like Receptor-Mediated Signaling on Expression of Protein S and C4b-Binding Protein in the Liver

Directory of Open Access Journals (Sweden)

Tatsuya Hayashi

2010-01-01

Full Text Available Protein S (PS, mainly synthesized in hepatocytes and endothelial cells, plays a critical role as a cofactor of anticoagulant activated protein C (APC. PS activity is regulated by C4b-binding protein (C4BP, structurally composed of seven α-chains (C4BPα and a β-chain (C4BPβ. In this paper, based primarily on our previous studies, we review the lipopolysaccharide (LPS-induced signaling which affects expression of PS and C4BP in the liver. Our in vivo studies in rats showed that after LPS injection, plasma PS levels are significantly decreased, whereas plasma C4BP levels first are transiently decreased after 2 to 12 hours and then significantly increased after 24 hours. LPS decreases PS antigen and mRNA levels in both hepatocytes and sinusoidal endothelial cells (SECs, and decreases C4BP antigen and both C4BPα and C4BPβ mRNA levels in hepatocytes. Antirat CD14 and antirat Toll-like receptor (TLR-4 antibodies inhibited LPS-induced NFκB activation in both hepatocytes and SECs. Furthermore, inhibitors of NFκB and MEK recovered the LPS-induced decreased expression of PS in both cell types and the LPS-induced decreased expression of C4BP in hepatocytes. These data suggest that the LPS-induced decrease in PS expression in hepatocytes and SECs and LPS-induced decrease in C4BP expression in hepatocytes are mediated by MEK/ERK signaling and NFκB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism.

Leukotriene B4 receptors on guinea pig alveolar eosinophils

International Nuclear Information System (INIS)

Maghni, K.; de Brum-Fernandes, A.J.; Foeldes-Filep, E.G.; Gaudry, M.; Borgeat, P.; Sirois, P.

1991-01-01

The existence of receptors for LTB4 on highly purified guinea pig alveolar eosinophils was investigated. Massive infiltration of eosinophils in alveolar spaces was induced in guinea pigs by i.v. injections of Sephadex beads G50 (16 mg/kg). Alveolar eosinophils (50 x 10(6) cells) were purified to approximately 98% by Percoll continuous density gradient centrifugation. The binding studies indicated that alveolar eosinophils bind LTB4 in a saturable, reversible and specific manner. Scatchard analysis indicated the existence of high-affinity binding sites (Kd1 = 1.00 ± 0.22 nM; Bmax1 = 966 ± 266 sites/cell) and low-affinity binding sites (Kd2 = 62.5 ± 8.9 nM; Bmax2 = 5557 ± 757 sites/cell). The metabolism of LTB4 by alveolar eosinophils in binding conditions was assessed by RP-HPLC and no significant degradation of [3H]LTB4 was observed. LTB4 dose-dependently stimulated eosinophil migration in both chemokinesis and chemotaxis assays with an EC50 value of 1.30 ± 0.14 and 18.14 ± 1.57 nM, respectively. LTB4 caused a dose-dependent increase in the production of superoxide anion with an apparent EC50 value of 50 x 10(-9) M in the authors experimental conditions. LTB4 also induced a dose-dependent increase in the generation of TxA2 with an EC50 value of 46.2 x 10(-9) M. Taken together, their results demonstrated that guinea pig alveolar eosinophils express two classes of specific receptors for LTB4. The high-affinity binding sites seem associated to chemokinesis and chemotaxis whereas the low-affinity binding sites seem associated to superoxide anion production and generation of TxA2. The existence of LTB4 receptors in eosinophils could explain the presence of these cells in hypersensitivity reactions

Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity at Angiotensin II Type 1 Receptor and Peroxisome Proliferator-Activated Receptor-[gamma

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Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan; Bigge, Christopher F.; Chen, Jing; Davis, Jo Ann; Dudley, Danette A.; Edmunds, Jeremy J.; Esmaeil, Nadia; Geyer, Andrew; Heemstra, Ronald J.; Jalaie, Mehran; Ohren, Jeffrey F.; Ostroski, Robert; Ellis, Teresa; Schaum, Robert P.; Stoner, Chad (Pfizer)

2013-03-07

Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partial PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.

Involvement of Striatal Cholinergic Interneurons and M1 and M4 Muscarinic Receptors in Motor Symptoms of Parkinson's Disease.

Science.gov (United States)

Ztaou, Samira; Maurice, Nicolas; Camon, Jeremy; Guiraudie-Capraz, Gaëlle; Kerkerian-Le Goff, Lydia; Beurrier, Corinne; Liberge, Martine; Amalric, Marianne

2016-08-31

Over the last decade, striatal cholinergic interneurons (ChIs) have reemerged as key actors in the pathophysiology of basal-ganglia-related movement disorders. However, the mechanisms involved are still unclear. In this study, we address the role of ChI activity in the expression of parkinsonian-like motor deficits in a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion model using optogenetic and pharmacological approaches. Dorsal striatal photoinhibition of ChIs in lesioned ChAT(cre/cre) mice expressing halorhodopsin in ChIs reduces akinesia, bradykinesia, and sensorimotor neglect. Muscarinic acetylcholine receptor (mAChR) blockade by scopolamine produces similar anti-parkinsonian effects. To decipher which of the mAChR subtypes provides these beneficial effects, systemic and intrastriatal administration of the selective M1 and M4 mAChR antagonists telenzepine and tropicamide, respectively, were tested in the same model of Parkinson's disease. The two compounds alleviate 6-OHDA lesion-induced motor deficits. Telenzepine produces its beneficial effects by blocking postsynaptic M1 mAChRs expressed on medium spiny neurons (MSNs) at the origin of the indirect striatopallidal and direct striatonigral pathways. The anti-parkinsonian effects of tropicamide were almost completely abolished in mutant lesioned mice that lack M4 mAChRs specifically in dopamine D1-receptor-expressing neurons, suggesting that postsynaptic M4 mAChRs expressed on direct MSNs mediate the antiakinetic action of tropicamide. The present results show that altered cholinergic transmission via M1 and M4 mAChRs of the dorsal striatum plays a pivotal role in the occurrence of motor symptoms in Parkinson's disease. The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinson's disease. Here, using optogenetic and pharmacological approaches, we investigated the involvement of striatal

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

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Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

2015-01-01

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

CRF receptor antagonist astressin-B reverses and prevents alopecia in CRF over-expressing mice.

Directory of Open Access Journals (Sweden)

Lixin Wang

2011-02-01

Full Text Available Corticotropin-releasing factor (CRF signaling pathways are involved in the stress response, and there is growing evidence supporting hair growth inhibition of murine hair follicle in vivo upon stress exposure. We investigated whether the blockade of CRF receptors influences the development of hair loss in CRF over-expressing (OE-mice that display phenotypes of Cushing's syndrome and chronic stress, including alopecia. The non-selective CRF receptors antagonist, astressin-B (5 µg/mouse injected peripherally once a day for 5 days in 4-9 months old CRF-OE alopecic mice induced pigmentation and hair re-growth that was largely retained for over 4 months. In young CRF-OE mice, astressin-B prevented the development of alopecia that occurred in saline-treated mice. Histological examination indicated that alopecic CRF-OE mice had hair follicle atrophy and that astressin-B revived the hair follicle from the telogen to anagen phase. However, astressin-B did not show any effect on the elevated plasma corticosterone levels and the increased weights of adrenal glands and visceral fat in CRF-OE mice. The selective CRF₂ receptor antagonist, astressin₂-B had moderate effect on pigmentation, but not on hair re-growth. The commercial drug for alopecia, minoxidil only showed partial effect on hair re-growth. These data support the existence of a key molecular switching mechanism triggered by blocking peripheral CRF receptors with an antagonist to reset hair growth in a mouse model of alopecia associated with chronic stress.

Solubilization and cleavage of human neutrophil (N) affinity-labeled receptors for leukotriene B4 (LTB4)

International Nuclear Information System (INIS)

Marotti, T.; Young, R.N.; Gifford, L.A.; Goldman, D.W.; Goetzl, E.J.

1986-01-01

LTB 4 chemotactic receptors in purified N plasma membranes (PMs) have been affinity-labeled with [ 3 H]-C-1 aminopropylamide-LTB 4 ([ 3 H]APA-LTB 4 ) by disuccinimidyl suberate (DSS) cross-linking. Intact Ns were pretreated with diisopropylfluorophosphate, suspended at 10 7 /ml in Hanks' solution-10 mM HEPES (pH 7.4), incubated for 30 min at 4 0 C with 30 nM [ 3 H]APA-LTB 4 and 25 min with 1 mM bis[2-(succinimidooxycarbonyloxy)-ethyl] sulfone, an impermeant analog of DSS, and sonified for 30 sec at 4 0 C. The 10,000 g supernatant of the sonicate was centrifuged at 40,000 g for 30 min at 4 0 C on a discontinuous gradient of 10-50 g % sucrose, from which a mean of 78% of the radiolabel was recovered with PM markers. The extent and specificity of labeling of intact N receptors were similar to those of receptors in PMs. The radioactively-labeled receptors appeared as a single band of 35-40 kd in sodium dodecyl sulfate (SDS) 10 g % polyacrylamide gel electrophoresis. Cleavage of radiolabeled receptors with 1 mg/ml of cyanogen bromide in 70% formic acid for 18 hr at room temperature or with 30 mM HCl under N 2 for 4 hr at 105 0 C converted a mean of 18-32% of the radioactivity to a band of 14 kd in SDS-15 g % PAGE. N receptors for LTB 4 , thus, are localized in the PM and can be isolated for structural studies

Involvement of the histamine H{sub 4} receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

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Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake [Division of Clinical Sciences and Neuropsychopharmacology, Faculty and Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 (Japan); Ozaki, Norio [Department of Psychiatry, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Noda, Yukihiro, E-mail: ynoda@meijo-u.ac.jp [Division of Clinical Sciences and Neuropsychopharmacology, Faculty and Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 (Japan)

2016-09-01

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100 μM) and doxorubicin (0.2 µM) decreased the cell survival rate, but olanzapine (1–100 µM) did not. Under granulocytic differentiation for 5 days, clozapine, even at a concentration of 25 μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H{sub 4} receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H{sub 4} receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H{sub 4} receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H{sub 4} receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation

EphB4 localises to the nucleus of prostate cancer cells

International Nuclear Information System (INIS)

Mertens-Walker, Inga; Lisle, Jessica E.; Nyberg, William A.; Stephens, Carson R.; Burke, Leslie; Rutkowski, Raphael; Herington, Adrian C.; Stephenson, Sally-Anne

2015-01-01

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation

EphB4 localises to the nucleus of prostate cancer cells

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Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Lisle, Jessica E. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Nyberg, William A. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Stephens, Carson R. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Burke, Leslie [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Rutkowski, Raphael; Herington, Adrian C.; Stephenson, Sally-Anne [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia)

2015-04-10

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.

Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Science.gov (United States)

Yang, Jinju; Qin, Nannan; Zhang, Hongwei; Yang, Rui; Xiang, Benqiong; Wei, Qun

2016-04-19

Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

Bradykinin B2 receptor expression in the bronchial mucosa of allergic asthmatics: the role of NF-kB

NARCIS (Netherlands)

Ricciardolo, F. L. M.; Petecchia, L.; Sorbello, V.; Di Stefano, A.; Usai, C.; Massaglia, G. M.; Gnemmi, I.; Mognetti, B.; Hiemstra, P. S.; Sterk, P. J.; Sabatini, F.

2016-01-01

Bradykinin (BK) mediates acute allergic asthma and airway remodelling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study, B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after

The superfamily of C3b/C4b-binding proteins

DEFF Research Database (Denmark)

Kristensen, Torsten; D'Eustachio, P; Ogata, R T

1987-01-01

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing...... at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain...... of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively...

Molecular Interactions between NR4A Orphan Nuclear Receptors and NF-κB Are Required for Appropriate Inflammatory Responses and Immune Cell Homeostasis.

Science.gov (United States)

Murphy, Evelyn P; Crean, Daniel

2015-06-29

Appropriate innate and adaptive immune responses are essential for protection and resolution against chemical, physical or biological insults. Immune cell polarization is fundamental in orchestrating distinct phases of inflammation, specifically acute phase responses followed by resolution and tissue repair. Dysregulation of immune cell and inflammatory responses is a hallmark of multiple diseases encompassing atherosclerosis, rheumatoid arthritis, psoriasis and metabolic syndromes. A master transcriptional mediator of diverse inflammatory signaling and immune cell function is NF-κB, and altered control of this key regulator can lead to an effective switch from acute to chronic inflammatory responses. Members of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors crosstalk with NF-κB to regulate immune cell function(s). Within the NR superfamily the NR4A1-3 orphan receptors have emerged as important regulators of immune cell polarization and NF-κB signaling. NR4A receptors modulate NF-κB activity in a dynamic fashion, either repressing or enhancing target gene expression leading to altered inflammatory outcome. Here we will discuss the pivotal role NR4A's receptors play in orchestrating immune cell homeostasis through molecular crosstalk with NF-κB. Specifically, we will examine such NR4A/NF-κB interactions within the context of distinct cell phenotypes, including monocyte, macrophage, T cells, endothelial, and mesenchymal cells, which play a role in inflammation-associated disease. Finally, we review the therapeutic potential of altering NR4A/NF-κB interactions to limit hyper-inflammatory responses in vivo.

Possible Involvement of Human Mast Cells in the Establishment of Pregnancy via Killer Cell Ig-Like Receptor 2DL4.

Science.gov (United States)

Ueshima, Chiyuki; Kataoka, Tatsuki R; Hirata, Masahiro; Sugimoto, Akihiko; Iemura, Yoshiki; Minamiguchi, Sachiko; Nomura, Takashi; Haga, Hironori

2018-06-01

The involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Role of ErbB receptors in cancer cell migration and invasion

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Aline eAppert-Collin

2015-11-01

Full Text Available Growth factors mediate their diverse biologic responses (regulation of cellular proliferation, differentiation, migration and survival by binding to and activating cell-surface receptors with intrinsic protein kinase activity named Receptor Tyrosine Kinases (RTKs. About 60 RTKs have been identified and can be classified into more than 16 different receptor families. Their activity is normally tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in alterations in the physiological activities of cells. The ErbB receptor family of RTKs comprises four distinct receptors: the EGFR (also known as ErbB1/HER1, ErbB2 (neu, HER2, ErbB3 (HER3 and ErbB4 (HER4. ErbB family members are often overexpressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. EGFR has been found to be amplified in gliomas and non-small-cell lung carcinoma while ErbB2 amplifications are seen in breast, ovarian, bladder, non-small-cell lung carcinoma, as well as several other tumor types. Several data have shown that ErbB receptor family and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion by modulating extracellular matrix components. Recent findings indicate that extracellular matrix components such as matrikines bind specifically to EGF receptor and promote cell invasion. In this review, we will present an in-depth overview of the structure, mechanisms, cell signaling, and functions of ErbB family receptors in cell adhesion and migration. Furthermore, we will describe in a last part the new strategies developed in anti-cancer therapy to inhibit ErbB family receptor activation.

Liver X receptor α and farnesoid X receptor are major transcriptional regulators of OATP1B1.

Science.gov (United States)

Meyer Zu Schwabedissen, Henriette E; Böttcher, Kerstin; Chaudhry, Amarjit; Kroemer, Heyo K; Schuetz, Erin G; Kim, Richard B

2010-11-01

Organic anion transporting polypeptide 1B1 (OATP1B1) is a liver-enriched transporter involved in the hepatocellular uptake of many endogenous molecules and several structurally divergent drugs in clinical use. Although OATP1B1 coding region polymorphisms are known to make an impact on substrate drug disposition in humans, little is known regarding the mechanisms underlying the transcriptional regulation of this transporter. In this study, we note that messenger RNA (mRNA) expression of OATP1B1 in a large human liver bank exhibited marked interindividual variability that was not associated with coding region polymorphisms. Accordingly, we hypothesized that such variability in expression is reflective of nuclear receptor-mediated transcriptional regulation of this transporter. We tested prototypical ligands for the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) α, and farnesoid X receptor (FXR) in a human hepatoma-derived cell line and noted induction of OATP1B1 mRNA when the cells were treated with LXRα or FXR ligands. To confirm a direct role for LXRα and FXR to OATP1B1 expression, we performed detailed promoter analysis and cell-based reporter gene assays resulting in the identification of two functional FXR response elements and one LXRα response element. The direct interaction between nuclear receptors with the identified response elements was assessed using chromatin immunoprecipitation assays. Using isolated primary human hepatocytes, we show that LXRα or FXR agonists, but not PXR or CAR agonists, are capable of OATP1B1 induction. We note that OATP1B1 transcriptional regulation is under dual nuclear receptor control through the oxysterol sensing LXRα and the bile acid sensor FXR. Accordingly, the interplay between OATP1B1 and nuclear receptors may play an important and heretofore unrecognized role during cholestasis, drug-induced liver injury, and OATP1B1 induction-related drug interactions.

Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage.

Science.gov (United States)

Zhang, Yan; Igwe, Orisa J

2018-01-01

Disturbances in redox equilibrium in tissue can lead to inflammatory state, which is a mediatory factor in many human diseases. The mechanism(s) by which exogenous oxidants may activate an inflammatory response is not fully understood. Emerging evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a major role in "sterile" inflammation. In the present study, we used murine macrophage RAW-Blue cells, which are chromosomally integrated with secreted embryonic alkaline phosphatase (SEAP) inducible by NF-κB. We confirmed the expression of TLR4 mRNA and protein in RAW-Blue cells by RT-PCR and Western blot, respectively. We showed that oxidants increased intracellular reactive oxygen species production and lipid peroxidation, which resulted in decreased intracellular total antioxidant capacity. Consistent with the actions of TLR4-specific agonist LPS-EK, exogenous oxidants increased transcriptional activity of NF-κB p65 with subsequent release of NF-κB reporter gene SEAP. These effects were blocked by pretreatment with TLR4 neutralizing pAb and TLR4 signaling inhibitor CLI-095. In addition, oxidants decreased the expression of IκBα with enhanced phosphorylation at the Tyr42 residue. Finally, oxidants and LPS-EK increased TNFα production, but did not affect IL-10 production, which may cause imbalance between pro- and anti-inflammatory processes, which CLI-095 inhibited. For biological relevance, we confirmed that oxidants increased release of TNFα and IL-6 in primary macrophages derived from TLR4-WT and TLR4-KO mice. Our results support the involvement of TLR4 mediated oxidant-induced inflammatory phenotype through NF-κB activation in macrophages. Thus exogenous oxidants may play a role in activating inflammatory phenotypes that propagate and maintain chronic disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model

Energy Technology Data Exchange (ETDEWEB)

Liu, Yu-Ching [Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan (China); Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Ho, Heng-Chien; Lee, Miau-Rong [Department of Biochemistry, China Medical University, Taichung 404, Taiwan (China); Lai, Kuang-Chi [Department of Surgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan (China); School of Medicine, China Medical University, Taichung 404, Taiwan (China); Yeh, Chung-Min; Lin, Yueh-Min [Department of Pathology, Changhua Christian Hospital, Changhua 500, Taiwan (China); Ho, Tin-Yun [School of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Hsiang, Chien-Yun, E-mail: cyhsiang@mail.cmu.edu.tw [Department of Microbiology, China Medical University, Taichung 404, Taiwan (China); Chung, Jing-Gung, E-mail: jgchung@mail.cmu.edu.tw [Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan (China); Department of Biotechnology, Asia University, Taichung 413, Taiwan (China)

2012-07-15

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9–18 fold) of induction in the microarray data, and its early induction was observed in a 2 h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model

International Nuclear Information System (INIS)

Liu, Yu-Ching; Ho, Heng-Chien; Lee, Miau-Rong; Lai, Kuang-Chi; Yeh, Chung-Min; Lin, Yueh-Min; Ho, Tin-Yun; Hsiang, Chien-Yun; Chung, Jing-Gung

2012-01-01

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9–18 fold) of induction in the microarray data, and its early induction was observed in a 2 h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

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Mattias N E Forsell

2008-10-01

Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

Science.gov (United States)

Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

2008-10-03

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

Toll-like receptors in the pathogenesis of human B cell malignancies

NARCIS (Netherlands)

Isaza-Correa, Johana M.; Liang, Zheng; van den Berg, Anke; Diepstra, Arjan; Visser, Lydia

2014-01-01

Toll-like receptors (TLRs) are important players in B-cell activation, maturation and memory and may be involved in the pathogenesis of B-cell lymphomas. Accumulating studies show differential expression in this heterogeneous group of cancers. Stimulation with TLR specific ligands, or agonists of

Radiosynthesis of 3-{l_brace}[4-(4-[{sup 18}F]fluorobenzyl)]piperazin-1-yl{r_brace}methyl-1H-pyrrolo[2,3-b]pyridine: A potential dopamine D{sub 4} receptor imaging agent

Energy Technology Data Exchange (ETDEWEB)

Haibin Tian; Duanzhi Yin; Junling Li; Lan Zhang; Cunfu Zhang; Yongxian Wang; Wei Zhou [Radiopharmaceutical Research Center, Shanghai Inst. of Nuclear Research, The Chinese Academy of Sciences, Shanghai, SH (China)

2003-07-01

The dopamine D{sub 4} receptor (D{sub 4}R) is expressed in low density in various extrastriatal brain regions. This receptor subtype is discussed in relation to the pathophysiology and treatment of schizophrenia but to date no selective positron emission tomography (PET) ligand is available to study its distribution in vivo. The 7-azaindole derivative 3-([4-(4-iodophenyl)piperazin-1-yl]-methyl)-1H-pyrrolo [2,3-b]pyridine (L-750,667) is a novel, high-affinity (K{sub i}=0.51nM) and selective D{sub 4}R ligand. L-750,667 analogue 3-[4-(4-[{sup 18}F]fluorobenzyl)]piperazin-1-yl methyl-1H-pyrrolo[2,3-b]-pyridine was prepared by reacting 3-(piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine with 4-[ 18F]fluorobenzaldehyde, which was labeled with no carrier added [ 18F]fluoride. The radiochemical yield of 3-[4-(4-[{sup 18}F]fluorobenzyl)]piperazin-1-yl methyl-1H-pyrrolo[2,3-b]pyridine was 12.0% at end of synthesis (EOS), and the synthesis time was 73min. The labeled benzaldehydes may be useful precursors for the radiosyntheses of other complex radiotracers for PET.

5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

DEFF Research Database (Denmark)

Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

2006-01-01

39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

Antidepressants inhibit P2X4 receptor function: a possible involvement in neuropathic pain relief

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Tozaki-Saitoh Hidetoshi

2009-04-01

Full Text Available Abstract Background Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X4 receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X4 receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X4 receptors and analysed their analgesic mechanism using an animal model of neuropathic pain. Results Antidepressants strongly inhibited ATP-mediated Ca2+ responses in P2X4 receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X4 receptors, with IC50 values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine. Conclusion These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X4 receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part

(3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

International Nuclear Information System (INIS)

Cheng, J.B.; Kohi, F.; Townley, R.G.

1986-01-01

To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Directory of Open Access Journals (Sweden)

Yingying Cai

Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Science.gov (United States)

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Signal transduction through the IL-4 and insulin receptor families.

Science.gov (United States)

Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

1995-07-01

Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

SR-B1 Is a Silica Receptor that Mediates Canonical Inflammasome Activation

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Misato Tsugita

2017-01-01

Full Text Available The inhalation of silica dust is associated with fibrosis and lung cancer, which are triggered by macrophage inflammatory responses; however, how macrophages recognize silica remains largely unknown. Here, we identify by functional expression cloning the class B scavenger receptor SR-B1 as a silica receptor. Through an extracellular α-helix, both mouse and human SR-B1 specifically recognized amorphous and crystalline silica, but not titanium dioxide nanoparticles, latex nanoparticles, or monosodium urate crystals, although all particles exhibited negative surface potentials. Genetic deletion of SR-B1 and masking of SR-B1 by monoclonal antibodies showed that SR-B1-mediated recognition of silica is associated with caspase-1-mediated inflammatory responses in mouse macrophages and human peripheral blood monocytes. Furthermore, SR-B1 was involved in silica-induced pulmonary inflammation in mice. These results indicate that SR-B1 is a silica receptor associated with canonical inflammasome activation.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

International Nuclear Information System (INIS)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue; Tsuchihashi, Kenji; Ueda, Shiho; Masuko, Takashi; Saya, Hideyuki; Nagano, Osamu

2016-01-01

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Kinin B1 receptors contributes to acute pain following minor surgery in humans

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Brahim Jaime S

2010-02-01

Full Text Available Abstract Background Kinins play an important role in regulation of pain and hyperalgesia after tissue injury and inflammation by activating two types of G-protein-coupled receptors, the kinin B1 and B2 receptors. It is generally accepted that the B2 receptor is constitutively expressed, whereas the B1 receptor is induced in response to inflammation. However, little is known about the regulatory effects of kinin receptors on the onset of acute inflammation and inflammatory pain in humans. The present study investigated the changes in gene expression of kinin receptors and the levels of their endogenous ligands at an early time point following tissue injury and their relation to clinical pain, as well as the effect of COX-inhibition on their expression levels. Results Tissue injury resulted in a significant up-regulation in the gene expression of B1 and B2 receptors at 3 hours post-surgery, the onset of acute inflammatory pain. Interestingly, the up-regulation in the gene expression of B1 and B2 receptors was positively correlated to pain intensity only after ketorolac treatment, signifying an interaction between prostaglandins and kinins in the inflammatory pain process. Further, the gene expression of both B1 and B2 receptors were correlated. Following tissue injury, B1 ligands des-Arg9-BK and des-Arg10-KD were significantly lower at the third hour compared to the first 2 hours in both the placebo and the ketorolac treatment groups but did not differ significantly between groups. Tissue injury also resulted in the down-regulation of TRPV1 gene expression at 3 hours post-surgery with no significant effect by ketorolac treatment. Interestingly, the change in gene expression of TRPV1 was correlated to the change in gene expression of B1 receptor but not B2 receptor. Conclusions These results provide evidence at the transcriptional level in a clinical model of tissue injury that up-regulation of kinin receptors are involved in the development of the

The macrophage A2B adenosine receptor regulates tissue insulin sensitivity.

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Hillary Johnston-Cox

Full Text Available High fat diet (HFD-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR, an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR null background. Reinstatement of macrophage A2bAR expression in A2bAR null mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice.

P2X4: A fast and sensitive purinergic receptor

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Jaanus Suurväli

2017-10-01

Full Text Available Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations, about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly

Selective up-regulation of 5-HT(1B/1D) receptors during organ culture of cerebral arteries

DEFF Research Database (Denmark)

Hoel, N L; Hansen-Schwartz, J; Edvinsson, L

2001-01-01

5-Hydroxytryptamine (5-HT) is thought to be involved in migraine headache and the pathophysiology of cerebrovascular diseases. Previous data show that organ culture induces a phenotypic change in cerebral vessels. Therefore we investigated if these changes also applied for the vasoconstrictive 5-HT......(cultured) 6.8+/-0.4). The response was inhibited by the 5-HT(1B/1D) selective antagonist GR55562 (pEC50(fresh) 5.1+/-0.2 and pEC50(cultured) 6.0+/-0.3). The organ model might mimic the phenotypic changes during cerebrovascular diseases....... receptors. Rat cerebral arteries express 5-HT2 receptors. Using organ culture we observed a phenotypic change with a selective up-regulation of 5-HT(1B/1D) receptors. This was revealed by an increased sensitivity to the selective 5-HT(1B/1D) agonist 5-CT after organ culture (pEC50(fresh) 5.6+/-0.2 and pEC50...

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

Science.gov (United States)

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Nuclear receptor 4a3 (nr4a3 regulates murine mast cell responses and granule content.

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Gianni Garcia-Faroldi

Full Text Available Nuclear receptor 4a3 (Nr4a3 is a transcription factor implicated in various settings such as vascular biology and inflammation. We have recently shown that mast cells dramatically upregulate Nuclear receptor 4a3 upon activation, and here we investigated the functional impact of Nuclear receptor 4a3 on mast cell responses. We show that Nuclear receptor 4a3 is involved in the regulation of cytokine/chemokine secretion in mast cells following activation via the high affinity IgE receptor. Moreover, Nuclear receptor 4a3 negatively affects the transcript and protein levels of mast cell tryptase as well as the mast cell's responsiveness to allergen. Together, these findings identify Nuclear receptor 4a3 as a novel regulator of mast cell function.

NR4A nuclear receptors are orphans but not lonesome

NARCIS (Netherlands)

Kurakula, Kondababu; Koenis, Duco S.; van Tiel, Claudia M.; de Vries, Carlie J. M.

2014-01-01

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that

Effects of a histamine H4 receptor antagonist on cisplatin-induced anorexia in mice.

Science.gov (United States)

Yamamoto, Kouichi; Okui, Rikuya; Yamatodani, Atsushi

2018-04-12

Cancer chemotherapy often induces gastrointestinal symptoms such as anorexia, nausea, and vomiting. Antiemetic agents are effective in inhibiting nausea and vomiting, but patients still experience anorexia. We previously reported that chemotherapeutic agent-induced anorexia is associated with an increase of inflammatory cytokines. Other studies also reported that antagonism of the histamine H 4 receptor is anti-inflammatory. In this study, we investigated the involvement of the H 4 receptor in the development of chemotherapy-induced anorexia in mice. Cisplatin-induced anorexia occurred within 24 h of its administration and continued for 3 days. The early phase (day 1), but not the delayed phase (days 2 and 3), of anorexia was inhibited by the daily injection of a 5-HT 3 receptor antagonist (granisetron). However, a corticosteroid (dexamethasone) or selective H 4 receptor antagonist (JNJ7777120) abolished the delayed phases of anorexia. Cisplatin significantly increased TNF-α mRNA expression in the hypothalamus and spleen, and the period of expression increase paralleled the onset period of anorexia. In addition, pretreatment with JNJ7777120 completely inhibited the increased expression. These results suggest that TNF-α mRNA expression via H 4 receptors may contribute to the development of cisplatin-induced anorexia, and that H 4 receptor antagonists are potentially useful treatments. Copyright © 2018 Elsevier B.V. All rights reserved.

ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

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Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.; (UPENN-MED)

2009-09-25

The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

Cloning and expression of a rat brain α2B-adrenergic receptor

International Nuclear Information System (INIS)

Flordellis, C.S.; Handy, D.E.; Bresnahan, M.R.; Zannis, V.I.; Gavras, H.

1991-01-01

The authors isolated a cDNA clone (RBα 2B ) and its homologous gene (GRα 2B ) encoding an α 2B -adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (α 2 -C4) and divergent from the rat kidney nonglycosylated α 2B subtype (RNGα 2 ). Transient expression of RBα 2B in COS-7 cells resulted in high-affinity saturable binding for [ 3 H]rauwolscine and a high receptor number in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine > yohimbine > prazosin > oxymetazoline, with a prazosin-to-oxymetazoline K i ratio of 0.34. This profile is characteristic of the α 2B -adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major and two minor mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GRα 2B may be transcriptionally active. These findings show that rat brain expresses an α 2B -adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated α 2B subtype. Thus the rat expresses at least two divergent α 2B -adrenergic receptors

Peptides derived from the solvent-exposed loops 3 and 4 of BDNF bind TrkB and p75(NTR) receptors and stimulate neurite outgrowth and survival

DEFF Research Database (Denmark)

Fobian, Kristina; Owczarek, Sylwia; Budtz, Christian

2010-01-01

Brain-derived neurotrophic factor (BDNF) is critically involved in modeling the developing nervous system and is an important regulator of a variety of crucial functions in the mature CNS. BDNF exerts its action through interactions with two transmembrane receptors, either separately or in concert....... BDNF has been implicated in several neurological disorders, and irregularities in BDNF function may have severe consequences. Administration of BDNF as a drug has thus far yielded few practicable results, and the potential side effects when using a multifunctional protein are substantial. In an effort...... to produce more specific compounds without side effects, small peptides mimicking protein function have been developed. The present study characterized two mimetic peptides, Betrofin 3 and Betrofin 4, derived from the BDNF sequence. Both Betrofins bound the cognate BDNF receptors, TrkB and p75(NTR...

Tyrosine receptor kinase B receptor activation reverses the impairing effects of acute nicotine on contextual fear extinction.

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Kutlu, Munir Gunes; Cole, Robert D; Connor, David A; Natwora, Brendan; Gould, Thomas J

2018-03-01

Anxiety and stress disorders have been linked to deficits in fear extinction. Our laboratory and others have demonstrated that acute nicotine impairs contextual fear extinction, suggesting that nicotine exposure may have negative effects on anxiety and stress disorder symptomatology. However, the neurobiological mechanisms underlying the acute nicotine-induced impairment of contextual fear extinction are unknown. Therefore, based on the previous studies showing that brain-derived neurotrophic factor is central for fear extinction learning and acute nicotine dysregulates brain-derived neurotrophic factor signaling, we hypothesized that the nicotine-induced impairment of contextual fear extinction may involve changes in tyrosine receptor kinase B signaling. To test this hypothesis, we systemically, intraperitoneally, injected C57BL/6J mice sub-threshold doses (2.5 and 4.0 mg/kg) of 7,8-dihydroxyflavone, a small-molecule tyrosine receptor kinase B agonist that fully mimics the effects of brain-derived neurotrophic factor, or vehicle an hour before each contextual fear extinction session. Mice also received injections, intraperitoneally, of acute nicotine (0.18 mg/kg) or saline 2-4 min before extinction sessions. While the animals that received only 7,8-dihydroxyflavone did not show any changes in contextual fear extinction, 4.0 mg/kg of 7,8-dihydroxyflavone ameliorated the extinction deficits in mice administered acute nicotine. Overall, these results suggest that acute nicotine-induced impairment of context extinction may be related to a disrupted brain-derived neurotrophic factor signaling.

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

International Nuclear Information System (INIS)

Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo; Mukdsi, Jorge Humberto; Mascanfroni, Iván Darío; Pellizas, Claudia Gabriela; Gutiérrez, Silvina; Torres, Alicia Inés; De Paul, Ana Lucía

2013-01-01

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

Energy Technology Data Exchange (ETDEWEB)

Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo; Mukdsi, Jorge Humberto [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina); Mascanfroni, Iván Darío; Pellizas, Claudia Gabriela [Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Av. Haya de la Torre y Medina Allende, Ciudad Universitaria, CP 5000, Córdoba (Argentina); Gutiérrez, Silvina; Torres, Alicia Inés [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina); De Paul, Ana Lucía, E-mail: adepaul@cmefcm.uncor.edu [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina)

2013-11-15

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K

Implication of 5-HT(2B) receptors in the serotonin syndrome.

Science.gov (United States)

Diaz, Silvina Laura; Maroteaux, Luc

2011-09-01

The serotonin (5-HT) syndrome occurs in humans after antidepressant overdose or combination of drugs inducing a massive increase in extracellular 5-HT. Several 5-HT receptors are known to participate in this syndrome in humans and animal models. The 5-HT(2B) receptor has been proposed as a positive modulator of serotonergic activity, but whether it is involved in 5-HT syndrome has not yet been studied. We analyzed here, a putative role of 5-HT(2B) receptors in this disorder by forced swimming test (FST) and behavioral assessment in the open field. In FST, genetic (5-HT(2B)(-/-) mice) or pharmacological (antagonist RS127445 at 0.5 mg/kg) ablation of 5-HT(2B) receptors facilitated selective 5-HT reuptake inhibitors (SSRI)-induced increase of immobility time as well as expression of other symptoms related to 5-HT syndrome like hind limb abduction and Straub tail. Increase in immobility was also developed in FST by both wild type (WT) and 5-HT(2B)(-/-) mice after the administration of 5-HT(1A), 5-HT(2A) or 5-HT(2C) receptor agonists, 8-OH-DPAT (5 mg/kg), DOI (1 mg/kg), or WAY161503 (5 mg/kg), respectively. In contrast, the 5-HT(2B) receptor agonist BW723C86 (3 mg/kg) or 5-HT(1B) receptor agonist CGS12066A (2 mg/kg) decreased immobility time in both genotypes. The 5-HT syndrome induced by fluoxetine at high doses was blocked in WT and 5-HT(2B)(-/-) mice by administration of 5-HT(1A) and 5-HT(2C) receptor antagonists (WAY100635 0.5 mg/kg and SB242084 0.5 mg/kg) but not by the 5-HT(2A) receptor antagonist MDL100907 (1 mg/kg). By behavioral assessment, we confirmed that 5-HT(2B)(-/-) mice were more prone to develop 5-HT syndrome symptoms after administration of high dose of SSRIs or the 5-HT precursor 5-Hydroxytryptophan, 5-HTP, even if increases in 5-HT plasma levels were similar in both genotypes. This evidence suggests that the presence of 5-HT(2B) receptors hinders acute 5-HT toxicity once high levels of 5-HT are attained. Therefore, differential agonism

Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth.

Science.gov (United States)

Soong, Joanne; Chen, Yulin; Shustef, Elina M; Scott, Glynis A

2012-04-01

Semaphorins are secreted and membrane-bound proteins involved in neural pathfinding, organogenesis, and tumor progression, through Plexin and neuropilin receptors. We recently reported that Plexin B1, the Semaphorin 4D (Sema4D) receptor, is a tumor-suppressor protein for melanoma, which functions, in part, through inhibition of the oncogenic c-Met tyrosine kinase receptor. In this report, we show that Sema4D is a protective paracrine factor for normal human melanocyte survival in response to UV irradiation, and that it stimulates proliferation and regulates the activity of the c-Met receptor. c-Met receptor signaling stimulates melanocyte migration, partly through downregulation of the cell adhesion molecule E-cadherin. Sema4D suppressed activation of c-Met in response to its ligand, hepatocyte growth factor (HGF), and partially blocked the suppressive effects of HGF on E-cadherin expression in melanocytes and HGF-dependent migration. These data demonstrate a role for Plexin B1 in maintenance of melanocyte survival and proliferation in the skin, and suggest that Sema4D and Plexin B1 act cooperatively with HGF and c-Met to regulate c-Met-dependent effects in human melanocytes. Because our data show that Plexin B1 is profoundly downregulated by UVB in melanocytes, loss of Plexin B1 may accentuate HGF-dependent effects on melanocytes, including melanocyte migration.

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

Science.gov (United States)

Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

2014-07-01

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

Pharmacological characterization of homobaclofen on wild type and mutant GABA(B)1b receptors coexpressed with the GABA(B)2 receptor

DEFF Research Database (Denmark)

Jensen, Anders A.; Madsen, Bo E.; Krogsgaard-Larsen, P

2001-01-01

homogenate and in an assay of electrically induced contractions of guinea pig ileum. The results from the two tissues did, however, not correlate very well, and in order to further investigate these discrepancies, we have pharmacologically characterized these enantiomers on recombinant wild type and mutant...... rat GABA(B)1b receptors coexpressed with rat GABA(B)2 receptors. The results from this study correlate nicely with the binding data from rat brain. (R)-Homobaclofen was shown to act like (R)-baclofen albeit with 20-fold less potency, and (S)-homobaclofen was inactive on the receptor. The discrepancies...

The role of scavenger receptor B1 in infection with Mycobacterium tuberculosis in a murine model.

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Georgia Schäfer

2009-12-01

Full Text Available The interaction between Mycobacterium tuberculosis (Mtb and host cells is complex and far from being understood. The role of the different receptor(s implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1 as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1(-/- or wild type mice in vitro. Moreover, when wild type and SR-B1(-/- animals were infected with a low dose of Mtb (100 CFU/mouse there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNgamma, and IL10 were observed in SR-B1(-/- mice following infection with a high dose of Mtb (1000 CFU/mouse, which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.SR-B1 is involved in

Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

Energy Technology Data Exchange (ETDEWEB)

Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

2015-03-06

Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

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

Science.gov (United States)

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

2013-11-05

Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced. Copyright © 2013 Elsevier B.V. All rights reserved.

Facilitation of acetylcholine release in rat frontal cortex by indeloxazine hydrochloride: involvement of endogenous serotonin and 5-HT4 receptors.

Science.gov (United States)

Yamaguchi, T; Suzuki, M; Yamamoto, M

1997-12-01

Effects of indeloxazine hydrochloride, an inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake with a facilitatory effect on 5-HT release, on acetylcholine (ACh) output in frontal cortex of conscious rats were characterized using an in vivo microdialysis technique. Systemic administration of indeloxazine (3 and 10 mg/kg, i.p.) increased ACh and 5-HT output in a dose-dependent manner. Depletion of endogenous monoamines by reserpine and of 5-HT by p-chlorophenylalanine, but not that of catecholamines by alpha-methyl-p-tyrosine, significantly attenuated the facilitatory effect of indeloxazine on ACh release. When applied locally by reverse dialysis, indeloxazine (10 and 30 microM) and the selective 5-HT reuptake inhibitor citalopram (10 microM), but not the NE reuptake inhibitor maprotiline (30 microM), increased cortical ACh output. Indeloxazine (10 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT4 receptor antagonists RS23597 (50 microM) and GR113803 (1 microM), while the 5-HT1A antagonist WAY-100135 (100 microM), 5-HT1A/1B/beta-adrenoceptor antagonist (-)propranolol (150 microM), 5-HT2A/2C antagonist ritanserin (10 microM) and 5-HT3 antagonist ondansetron (10 microM) failed to significantly modify this effect. Neither depletion of monoamines nor treatment with serotonergic antagonists significantly changed the basal ACh level, indicating that endogenous monoamines do not tonically activate ACh release. These results suggest that indeloxazine-induced facilitation of ACh release in rat frontal cortex is mediated by endogenous 5-HT and involves at least in part cortical 5-HT4 receptors.

Bradykinin B2 receptor expression in the bronchial mucosa of allergic asthmatics: the role of NF-kB.

Science.gov (United States)

Ricciardolo, F L M; Petecchia, L; Sorbello, V; Di Stefano, A; Usai, C; Massaglia, G M; Gnemmi, I; Mognetti, B; Hiemstra, P S; Sterk, P J; Sabatini, F

2016-03-01

Bradykinin (BK) mediates acute allergic asthma and airway remodelling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study, B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls, examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analysed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 h after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was higher in asthmatics after diluent (B2R only) and allergen challenge than in controls (P kB (total and nuclear) increased after allergen compared with after diluent (P kB inhibitor (P kB expression. NF-kB inhibitor blocked IL-4/IL-13-induced increase in B2R expression in cultured fibroblasts, suggesting a role as potential anti-asthma drug. © 2015 John Wiley & Sons Ltd.

B-Cell Activation and Tolerance Mediated by B-Cell Receptor, Toll-Like Receptor and Survival Signal Crosstalk in SLE Pathogenesis

Science.gov (United States)

2015-10-01

H. Wagner, K. Takeda, and S. Akira. 2000 . A Toll-like receptor recognizes bacterial DNA. Nature 408: 740–745. 2. Kawai, T., and S. Akira. 2010. The...I. M. Carr , J. C. Fuller, R. M. Jackson, T. Lamb, T. A. Briggs, et al. 2009. Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as...Moreover, recent studies from Riley and colleagues (27) suggest that ABCs negatively influence B-lineage commitment or development of bone marrow

Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

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Barwell, James; Gingell, Joseph J; Watkins, Harriet A; Archbold, Julia K; Poyner, David R; Hay, Debbie L

2012-05-01

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

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Koch, Alexander W; Mathivet, Thomas; Larrivée, Bruno; Tong, Raymond K; Kowalski, Joe; Pibouin-Fragner, Laurence; Bouvrée, Karine; Stawicki, Scott; Nicholes, Katrina; Rathore, Nisha; Scales, Suzie J; Luis, Elizabeth; del Toro, Raquel; Freitas, Catarina; Bréant, Christiane; Michaud, Annie; Corvol, Pierre; Thomas, Jean-Léon; Wu, Yan; Peale, Franklin; Watts, Ryan J; Tessier-Lavigne, Marc; Bagri, Anil; Eichmann, Anne

2011-01-18

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature. Copyright © 2011 Elsevier Inc. All rights reserved.

Association of low-affinity FC gamma receptor 3B (FCGR3B) copy number variation with rheumatoid arthritis in Caucasian subjects

NARCIS (Netherlands)

Merriman, T.R.; Fanciulli, M.; Merriman, M.E.; Alizadeh, B.Z.; Koeleman, B.P.C.; Dalbeth, N.; Gow, P.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Vyse, T.; Aitman, T.; Radstake, T.; McKinney, C.

2009-01-01

Aim: There is increasing evidence that gene copy-number variation influences phenotypic variation. The low-affinity Fc receptor 3B (FCGR3B) is a copy-number polymorphic gene involved in the recruitment to sites of inflammation and activation of polymorphonuclear neutrophils (PMN). Given the

Lipoprotein profiles in human heterozygote carriers of a functional mutation P297S in scavenger receptor class B1

NARCIS (Netherlands)

Ljunggren, Stefan A.; Levels, Johannes H. M.; Hovingh, Kees; Holleboom, Adriaan G.; Vergeer, Menno; Argyri, Letta; Gkolfinopoulou, Christina; Chroni, Angeliki; Sierts, Jeroen A.; Kastelein, John J.; Kuivenhoven, Jan Albert; Lindahl, Mats; Karlsson, Helen

2015-01-01

The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1(P297S) mutation are characterized by increased HDL cholesterol

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

Science.gov (United States)

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L; Burgdorf, Sven

2016-09-20

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

NMDA receptor adjusted co-administration of ecstasy and cannabinoid receptor-1 agonist in the amygdala via stimulation of BDNF/Trk-B/CREB pathway in adult male rats.

Science.gov (United States)

Ashabi, Ghorbangol; Sadat-Shirazi, Mitra-Sadat; Khalifeh, Solmaz; Elhampour, Laleh; Zarrindast, Mohammad-Reza

2017-04-01

Consumption of cannabinoid receptor-1 (CB-1) agonist such as cannabis is widely taken in 3,4- methylenedioxymethamphetamine (MDMA) or ecstasy users; it has been hypothesized that co-consumption of CB-1 agonist might protect neurons against MDMA toxicity. N-methyl-d-aspartate (NMDA) receptors regulate neuronal plasticity and firing rate in the brain through Tyrosine-kinase B (Trk-B) activation. The molecular and electrophysiological association among NMDA and MDMA/Arachidonylcyclopropylamide (ACPA, a selective CB-1 receptor agonist) co-consumption was not well-known. Here, neuronal spontaneous activity, Brain-derived neurotrophic factor (BDNF), Trk-B and cAMP response element binding protein (CREB) phosphorylation levels were recognized in ACPA and MDMA co-injected rats. Besides, we proved the role of NMDA receptor on MDMA and ACPA combination on neuronal spontaneous activity and Trk-B/BDNF pathway in the central amygdala (CeA). Male rats were anesthetized with intra-peritoneal injections of urethane; MDMA, D-2-amino-5-phosphonopentanoate (D-AP5, NMDA receptor antagonist) were injected into CeA. ACPA was administrated by intra-cerebroventricular injection. Thirty minutes following injections, neuronal firing rate was recorded from CeA. Two hours after drug injection, amygdala was collected from brain for molecular evaluations. Single administration of MDMA and/or ACPA reduced firing rates compared with sham group in the CeA dose-dependently. Injection of D-AP5, ACPA and MDMA reduced firing rate compared with sham group (P<0.001). Interestingly, injection of ACPA+MDMA enhanced BDNF, Trk-B and CREB phosphorylation compared with MDMA groups. D-AP5, ACPA and MDMA co-injection decreased BDNF, Trk-B and CREB phosphorylation levels compared with ACPA+MDMA in the amygdala (P<0.01). Probably, NMDA receptors are involved in the protective role of acute MDMA+ACPA co-injection via BDNF/Trk-B/CREB pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

International Nuclear Information System (INIS)

Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

Science.gov (United States)

Sgnaolin, V; Pereira, T C B; Bogo, M R; Zanin, R; Battastini, A M O; Morrone, F B; Campos, M M

2013-08-01

Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

GABA-B receptor activation and conflict behavior

International Nuclear Information System (INIS)

Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

1988-01-01

Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on [ 3 H]-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables

Social Context, Stress, Neuropsychiatric Disorders, and the Vasopressin 1b Receptor

Directory of Open Access Journals (Sweden)

Heather K. Caldwell

2017-10-01

Full Text Available The arginine vasopressin 1b receptor (Avpr1b is involved in the modulation of a variety of behaviors and is an important part of the mammalian hormonal stress axis. The Avpr1b is prominent in hippocampal CA2 pyramidal cells and in the anterior pituitary corticotrophs. Decades of research on this receptor has demonstrated its importance to the modulation of social recognition memory, social forms of aggression, and modulation of the hypothalamic-pituitary-adrenal axis, particularly under conditions of acute stress. Further, work in humans suggests that the Avpr1b may play a role in human neuropsychiatric disorders and its modulation may have therapeutic potential. This paper reviews what is known about the role of the Avpr1b in the context of social behaviors, the stress axis, and human neuropsychiatric disorders. Further, possible mechanisms for how Avpr1b activation within the hippocampus vs. Avpr1b activation within anterior pituitary may interact with one another to affect behavioral output are proposed.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury.

Science.gov (United States)

Huang, Rong-Shuang; Zhou, Jiao-Jiao; Feng, Yu-Ying; Shi, Min; Guo, Fan; Gou, Shen-Ju; Salerno, Stephen; Ma, Liang; Fu, Ping

2017-09-20

Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway. C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected. In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

HTLV-1 specific CD8+ T cell function augmented by blockade of 2B4/CD48 interaction in HTLV-1 infection.

Directory of Open Access Journals (Sweden)

Chibueze Chioma Ezinne

Full Text Available CD8+ T cell response is important in the response to viral infections; this response though is regulated by inhibitory receptors. Expression of inhibitory receptors has been positively correlated with CD8+ T cell exhaustion; the consequent effect of simultaneous blockade of these inhibitory receptors on CD8+ T cell response in viral infections have been studied, however, the role of individual blockade of receptor-ligand pair is unclear. 2B4/CD48 interaction is involved in CD8+T cell regulation, its signal transducer SAP (signaling lymphocyte activation molecule (SLAM-associated protein is required for stimulatory function of 2B4/CD244 on lymphocytes hence, we analyzed 2B4/CD244 (natural killer cell receptor and SAP (signaling lymphocyte activation molecule(SLAM-associated protein on total CD8+ and HTLV-1 specific CD8+T cells in HTLV-1 infection and the effect of blockade of interaction with ligand CD48 on HTLV-1 specific CD8+ T cell function. We observed a high expression of 2B4/CD244 on CD8+ T cells relative to uninfected and further upregulation on HTLV-1 specific CD8+ T cells. 2B4+ CD8+ T cells exhibited more of an effector and terminally differentiated memory phenotype. Blockade of 2B4/CD48 interaction resulted in improvement in function via perforin expression and degranulation as measured by CD107a surface mobilization on HTLV-1 specific CD8+ T cells. In the light of these findings, we thus propose an inhibitory role for 2B4/CD48 interaction on CD8+T cell function.

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Science.gov (United States)

Mechiche, Hakima; Grassin-Delyle, Stanislas; Robinet, Arnaud; Nazeyrollas, Pierre; Devillier, Philippe

2012-01-01

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD2 and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP1 and EP3 receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP1 antagonist). Butaprost (a selective prostanoid EP2 receptor agonist), misoprostol (a prostanoid EP2 and EP3 receptor agonist), 11-deoxy-PGE1 (a prostanoid EP2, EP3 and EP4 receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP1 receptors are involved in positive regulation of the beating rate. Prostanoid EP1 receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP1 and EP1 receptors (which positively regulate the spontaneous beating rate). PMID:22984630

Or47b receptor neurons mediate sociosexual interactions in the fruit fly Drosophila melanogaster.

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Lone, Shahnaz Rahman; Sharma, Vijay Kumar

2012-04-01

In the fruit fly Drosophila melanogaster, social interactions especially among heterosexual couples have been shown to have significant impact on the circadian timing system. Olfaction plays a major role in such interactions; however, we do not know yet specifically which receptor(s) are involved. Further, the role of circadian clock neurons in the rhythmic regulation of such sociosexual interactions (SSIs) is not fully understood. Here, we report the results of our study in which we assayed the locomotor activity and sleep-wake behaviors of male-male (MM), female-female (FF), and male-female (MF) couples from several wild-type and mutant strains of Drosophila with an aim to identify specific olfactory receptor(s) and circadian clock neurons involved in the rhythmic regulation of SSI. The results indicate that Or47b receptor neurons are necessary for SSI, as ablation or silencing of these neurons has a severe impact on SSI. Further, the neuropeptide pigment dispersing factor (PDF) and PDF-positive ventral lateral (LN(v)) clock neurons appear to be dispensable for the regulation of SSI; however, dorsal neurons may be involved.

Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

Science.gov (United States)

Luhrs, Lauren; Manlapaz, Cynthia; Kedzie, Karen; Rao, Sandhya; Cabrera-Ghayouri, Sara; Donello, John; Gil, Daniel

2016-12-17

Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and

Tritium-labelled leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site

International Nuclear Information System (INIS)

Cheng, J.B.; Cheng, E.I.; Kohi, F.; Townley, R.G.

1986-01-01

Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [ 3 H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [ 3 H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [ 3 H]LTB4 binding, but NaCl and KCl decreased it. Spleen [ 3 H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 0 C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[ 3 H]LTB4 competition study performed at 4 or 25 0 C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [ 3 H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [ 3 H]LTB4 binding

The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

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Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

2016-04-01

Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation.

Science.gov (United States)

Fujita, Masahide; Andoh, Tsugunobu; Saiki, Ikuo; Kuraishi, Yasushi

2008-02-01

We investigated whether endothelin (ET) would be involved in skin cancer pain in mice. Orthotopic inoculation of B16-BL6 melanoma cells into the plantar region of the hind paw produced marked mechanical allodynia in C57BL/6 mice. Intraplantar injections of the ET(A)-receptor antagonist BQ-123 (0.3 - 3 nmol/site), but not the ET(B)-receptor antagonist BQ-788 (1 and 3 nmol/site), inhibited mechanical allodynia in mice with grown melanoma. In naive mice, an intraplantar injection of tumor extract (1 and 3 mg/site), which was prepared from the grown melanoma in the paw, produced mechanical allodynia, which was inhibited by BQ-123 and BQ-788 at doses of 3 and 10 nmol/site. An intraplantar injection of ET-1 (1 and 10 pmol/site) elicited licking behavior, which was increased in the melanoma-bearing hind paw. BQ-123 (3 and 10 nmol/site) inhibited licking induced by ET-1 (10 pmol/site). The level of mRNA of ET(A), but not ET(B), receptor, was significantly increased in the dorsal root ganglia on the inoculated side. Cultured B16-BL6 cells contained ET, and the melanoma mass increased the concentration of ET as it grew bigger. These results suggest that ET-1 and ET(A) receptor are at least partly involved in the induction of pain induced by melanoma cell inoculation.

Involvement of transient receptor potential vanilloid 2 in intra-oral incisional pain.

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Urata, K; Shinoda, M; Ikutame, D; Iinuma, T; Iwata, K

2018-03-05

To examine whether transient receptor potential vanilloid 2 (TRPV2) contributes to the changes in intra-oral thermal and mechanical sensitivity following the incision of buccal mucosa. Buccal mucosal pain threshold was measured after the incision. Changes in the number of TRPV2-immunoreactive (IR) trigeminal ganglion (TG) neurons which innervate the whisker pad skin and buccal mucosa, changes in the number of isolectin B4-negative/isolectin B4-positive TRPV2-IR TG neurons which innervate the whisker pad skin and the buccal mucosa, and the effect of peripheral TRPV2 antagonism on the pain threshold of incisional whisker pad skin and buccal mucosa were examined after these injuries. Buccal mucosal pain hypersensitivities were induced on day 3 following the incision. The total number of TRPV2-IR TG neurons and the number of isolectin B4-negative TRPV2-IR TG neurons which innervate the whisker pad skin and buccal mucosa were increased. Buccal mucosal TRPV2 antagonism completely suppressed the heat and mechanical hypersensitivities, but not cold hypersensitivity. TRPV2 antagonist administration to the incisional whisker pad skin only partially suppressed pain hypersensitivities. The increased expression of TRPV2 in peptidergic TG neurons innervating the incisional buccal mucosa is predominantly involved in buccal mucosal heat hyperalgesia and mechanical allodynia following buccal mucosal incision. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

A new, highly selective CCK-B receptor radioligand ([3H][N-methyl-Nle28,31]CCK26-33): Evidence for CCK-B receptor heterogeneity

International Nuclear Information System (INIS)

Knapp, R.J.; Vaughn, L.K.; Fang, S.N.; Bogert, C.L.; Yamamura, M.S.; Hruby, V.J.; Yamamura, H.I.

1990-01-01

[N-methyl-Nle28,31]CCK26-33 (SNF 8702) is a nonsulfated cholecystokinin octapeptide analog that is highly selective for cholecystokinin-B (CCK-B) receptors. Inhibition studies using [125I] Bolton-Hunter-labeled CCK-8 show that SNF 8702 has over 4,000-fold greater affinity for CCK receptors in guinea pig cortex relative to those in guinea pig pancreas. SNF 8702 was tritium-labeled to a specific activity of 23.7 Ci/mmol and its binding properties characterized for guinea pig brain membrane preparations. [3H]SNF 8702 binds to a single site with high affinity (Kd = 0.69-0.90 nM) in guinea pig cortex, cerebellum, hippocampus and pons-medulla. Of these four tissues, the highest receptor density was measured in the cortex (86 fmol/mg of protein) and the lowest in the pons-medulla (22 fmol/mg of protein). In contrast to findings of single-site binding in some brain regions, evidence for CCK-B receptor heterogeneity is observed under other conditions. [3H]SNF 8702 binding to membranes prepared from whole guinea pig brain shows biphasic association kinetics at a concentration of 2.0 nM consistent with the presence of binding site heterogeneity. Binding site heterogeneity is consistently observed for [3H]SNF 8702 binding to guinea pig whole brain membranes in saturation studies where a high-affinity site (Kd = 0.31 nM) is distinguished from a low-affinity site (Kd = 3.3 nM). Binding site heterogeneity is also observed for the midbrain-thalamic region. CCK-B receptor heterogeneity is suggested by the effect of the guanyl nucleotide analogue, guanylyl-imidodiphosphate (Gpp(NH)p), on [3H]SNF 8702 binding to CCK-B receptors in the cerebellum

EphB4 promotes or suppresses Ras/MEK/ERK pathway in a context-dependent manner: Implications for EphB4 as a cancer target.

Science.gov (United States)

Xiao, Zhan; Carrasco, Rosa; Kinneer, Krista; Sabol, Darrin; Jallal, Bahija; Coats, Steve; Tice, David A

2012-06-01

EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4's function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.

Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats.

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Miszkiel, Joanna; Przegaliński, Edmund

2013-01-01

Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.

ERBB4 Mutations that Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19

Science.gov (United States)

Takahashi, Yuji; Fukuda, Yoko; Yoshimura, Jun; Toyoda, Atsushi; Kurppa, Kari; Moritoyo, Hiroyoko; Belzil, Veronique V.; Dion, Patrick A.; Higasa, Koichiro; Doi, Koichiro; Ishiura, Hiroyuki; Mitsui, Jun; Date, Hidetoshi; Ahsan, Budrul; Matsukawa, Takashi; Ichikawa, Yaeko; Moritoyo, Takashi; Ikoma, Mayumi; Hashimoto, Tsukasa; Kimura, Fumiharu; Murayama, Shigeo; Onodera, Osamu; Nishizawa, Masatoyo; Yoshida, Mari; Atsuta, Naoki; Sobue, Gen; Fifita, Jennifer A.; Williams, Kelly L.; Blair, Ian P.; Nicholson, Garth A.; Gonzalez-Perez, Paloma; Brown, Robert H.; Nomoto, Masahiro; Elenius, Klaus; Rouleau, Guy A.; Fujiyama, Asao; Morishita, Shinichi; Goto, Jun; Tsuji, Shoji

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3–5 years from onset. Familial ALS (FALS) comprises 5%–10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions. PMID:24119685

Does chronic nicotine alter neurotransmitter receptors involved in Parkinson's disease?

International Nuclear Information System (INIS)

Reilly, M.A.; Lapin, E.P.; Lajtha, A.; Maker, H.S.

1986-01-01

Cigarette smokers are fewer in number among Parkinson's Disease (PD) patients than among groups of persons who do not have PD. Several hypotheses have been proposed to explain this observation. One which must be tested is the possibility that some pharmacologic agent present in cigarette smoke may interact with some central nervous system component involved in PD. To this end, they have investigated the effect of chronic nicotine administration on receptors for some of the neurotransmitters that are affected in PD. Rats were injected for six weeks with saline or nicotine 0.8 mg/kg S.C., then killed and brains removed and dissected. The binding of ( 3 H)-ketanserin to serotonin receptors in frontal cortex and of ( 3 H)-domperidone to dopamine receptors in caudate was not affected. However, the binding of ( 3 H)-domperidone in nucleus accumbens was altered: the K/sub d/ increased from 0.16 +/- 0.02 nM to 0.61 +/- 0.07 nM, and the B/sub max/ increased from 507 +/- 47 fmol/mg protein to 910 +/- 43 fmol/mg (p < 0.001 for both comparisons). These values are based on three ligand concentrations. Additional studies are in progress to substantiate the data. It is concluded that chronic nicotine administration may alter dopamine receptors in nucleus accumbens

Purification of rat intestinal receptor for intrinsic factor-vitamin B12 complex

International Nuclear Information System (INIS)

Yamada, Shoji; Itaya, Harutaka; Nakazawa, Osamu; Fukuda, Morimichi.

1977-01-01

The intrinsic factor (IF) in a rat gastric mucosal extract was bound efficiently to vitamin B 12 -sepharose without significant change in its nature to produce IF-vitamin B 12 -sepharose. The purification of the intestinal receptor for the IF-vitamin B 12 complex was performed by the affinity chromatography using the IF-vitamin B 12 -sepharose as the affinity adsorbent. As a result of admixing the gastric mucosal extract sample with B 12 -sepharose while stirring for 4 hours, the adsorption was performed without any break through. Further, it was recognized that the B 12 -bound protein purified by the affinity chromatography using B 12 -sepharose was not much changed as compared with that before purification. Furthermore, it was recognized that IF-B 12 -sepharose was able to be made by binding IF with B 12 -sepharose which was made by coupling B 12 with the market-available AH-sepharose. The IF-B 12 -sepharose was washed with buffer solution, and then was loaded with the small intestine mucosal extract. Thereafter, the receptor was eluted by making di-valent cation inert with the buffer solution. After the removal of EDTA in the eluted solution by dialysis, the activity of the receptor was measured. 48.5% of the receptor activity loaded was recovered by the elution with EDTA. The specific activity of the receptor represented by the final amount of B 12 (pg)/the amount of protein (mg) in the purified substance was 335 folds of the original activity. (Iwakiri, K.)

Hypothalamic-pituitary thyroid axis alterations in female mice with deletion of the neuromedin B receptor gene.

Science.gov (United States)

Oliveira, Karen J; Paula, Gabriela S M; Império, Guinever E; Bressane, Nina O; Magalhães, Carolina M A; Miranda-Alves, Leandro; Ortiga-Carvalho, Tania M; Pazos-Moura, Carmen C

2014-11-01

Neuromedin B, a peptide highly expressed at the pituitary, has been shown to act as autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here we studied the thyroid axis of adult female mice lacking neuromedin B receptor (NBR-KO), compared to wild type (WT) littermates. They exhibited slight increase in serum TSH (18%), with normal pituitary expression of mRNA coding for α-glycoprotein subunit (Cga), but reduced TSH β-subunit mRNA (Tshb, 41%), lower intra-pituitary TSH content (24%) and increased thyroid hormone transporter MCT-8 (Slc16a2, 44%) and thyroid hormone receptor β mRNA expression (Thrb, 39%). NBR-KO mice exhibited normal thyroxine (T4) and reduced triiodothyronine (T3) (30%), with no alterations in the intra-thyroidal content of T4 and T3 or thyroid morphological changes. Hypothalamic thyrotropin-releasing hormone (TRH) mRNA (Trh) was increased (68%), concomitant with a reduction in type 2 deiodinase mRNA (Dio2, 30%) and no changes in MCT-8 and thyroid hormone receptor mRNA expression. NBR-KO mice exhibited a 56% higher increase in serum TSH in response to an acute single intraperitoneal injection of TRH concomitant with a non-significant increase in pituitary TRH receptor (Trhr) mRNA at basal state. The phenotype of female NBR-KO mice at the hypothalamus-pituitary axis revealed alterations in pituitary and hypothalamic gene expression, associated with reduced serum T3, and higher TSH response to TRH, with apparently normal thyroid morphology and hormonal production. Thus, results confirm that neuromedin B pathways are importantly involved in secretory pathways of TSH and revealed its participation in the in vivo regulation of gene expression of TSH β-subunit and pituitary MCT8 and Thrb and hypothalamic TRH and type 2 deiodinase. Copyright © 2014 Elsevier B.V. All rights reserved.

Hippocampal NMDA receptors are involved in rats' spontaneous object recognition only under high memory load condition.

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Sugita, Manami; Yamada, Kazuo; Iguchi, Natsumi; Ichitani, Yukio

2015-10-22

The possible involvement of hippocampal N-methyl-D-aspartate (NMDA) receptors in spontaneous object recognition was investigated in rats under different memory load conditions. We first estimated rats' object memory span using 3-5 objects in "Different Objects Task (DOT)" in order to confirm the highest memory load condition in object recognition memory. Rats were allowed to explore a field in which 3 (3-DOT), 4 (4-DOT), or 5 (5-DOT) different objects were presented. After a delay period, they were placed again in the same field in which one of the sample objects was replaced by another object, and their object exploration behavior was analyzed. Rats could differentiate the novel object from the familiar ones in 3-DOT and 4-DOT but not in 5-DOT, suggesting that rats' object memory span was about 4. Then, we examined the effects of hippocampal AP5 infusion on performance in both 2-DOT (2 different objects were used) and 4-DOT. The drug treatment before the sample phase impaired performance only in 4-DOT. These results suggest that hippocampal NMDA receptors play a critical role in spontaneous object recognition only when the memory load is high. Copyright © 2015 Elsevier B.V. All rights reserved.

Very late-onset group B Streptococcus meningitis, sepsis, and systemic shigellosis due to interleukin-1 receptor-associated kinase-4 deficiency.

Science.gov (United States)

Krause, Jens C; Ghandil, Pegah; Chrabieh, Maya; Casanova, Jean-Laurent; Picard, Capucine; Puel, Anne; Creech, C Buddy

2009-11-01

We describe a child with very late-onset group B Streptococcus sepsis and meningitis, systemic shigellosis, and chronic osteomyelitis. Peripheral blood cells obtained from the patient and her brother did not respond to stimulation with either interleukin-1beta or lipopolysaccharide. Sequencing of the interleukin-1 receptor-associated kinase-4 gene revealed 2 novel mutations.

The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

Science.gov (United States)

Keegan, A D; Pierce, J H

1994-02-01

Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

Science.gov (United States)

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

DEFF Research Database (Denmark)

Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav

2010-01-01

The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... constitutes part of the dimerization arm of ErbB3. Inherbin3 binds to the extracellular domains of all four ErbB receptors, with the lowest peptide binding affinity for ErbB4. Inherbin3 functions as an antagonist of epidermal growth factor (EGF)-ErbB1 signaling. We show that Inherbin3 inhibits EGF-induced Erb....... Structural studies have revealed that ErbB receptor dimers are stabilized by receptor-receptor interactions, primarily mediated by a region in the second extracellular domain, termed the "dimerization arm". The present study is the first biological characterization of a peptide, termed Inherbin3, which...

Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

Science.gov (United States)

Stoffels, Burkhard; Hupa, Kristof Johannes; Snoek, Susanne A; van Bree, Sjoerd; Stein, Kathy; Schwandt, Timo; Vilz, Tim O; Lysson, Mariola; Veer, Cornelis Van't; Kummer, Markus P; Hornung, Veit; Kalff, Joerg C; de Jonge, Wouter J; Wehner, Sven

2014-01-01

Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with

Dexmedetomidine reduces ventilator-induced lung injury (VILI by inhibiting Toll-like receptor 4 (TLR4/nuclear factor (NF-κB signaling pathway

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Hongli Chen

2018-02-01

Full Text Available Mechanical ventilation (MV may lead to ventilator-induced lung injury (VILI. Previous research has shown that dexmedetomidine attenuates pulmonary inflammation caused by MV, but the underlying mechanisms remain unclear. Our study aims to test whether dexmedetomidine has a protective effect against VILI and to explore the possible molecular mechanisms using the rat model. Thirty adult male Wistar rats weighing 200-250 g were randomly assigned to 5 groups (n = 6: control, low tidal volume MV (LMV, high tidal volume (HVT MV (HMV, HVT MV + dexmedetomidine (DEX, HVT MV + dexmedetomidine + yohimbine (DEX+Y. Rats were euthanized after being ventilated for 4 hours. Pathological changes, lung wet/dry (W/D weight ratio, lung myeloperoxidase (MPO activity, levels of inflammatory cytokines (i.e., interleukin [IL]-1β, tumor necrosis factor alpha [TNF-α], and IL-6 in the bronchoalveolar lavage fluid (BALF and lung tissues, expression of Toll-like receptor 4 (TLR4 and nuclear factor (NF-κB, and activation of NF-κB in lung tissues were measured. Compared with HMV, DEX group showed fewer pathological changes, lower W/D ratios and decreased MPO activity of the lung tissues and lower concentrations of the inflammatory cytokines in the BALF and lung tissues. Dexmedetomidine significantly inhibited the expression of TLR4 and NF-κB and activation of NF-κB. Yohimbine partly alleviated the effects of dexmedetomidine. Dexmedetomidine reduced the inflammatory response to HVT-MV and had a protective effect against VILI, with the inhibition of the TLR4/NF-κB signaling pathway, at least partly via α2-adrenoceptors.

SNAP23-Dependent Surface Translocation of Leukotriene B4 (LTB4) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB4.

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Min, Arim; Lee, Young Ah; Kim, Kyeong Ah; El-Benna, Jamel; Shin, Myeong Heon

2017-01-01

Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B 4 (LTB 4 ). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB 4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB 4 Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB 4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses. Copyright © 2016 American Society for Microbiology.

Expression of nicotinic acetylcholine receptors on human B-lymphoma cells

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Skok M. V.

2009-12-01

Full Text Available Aim. To find a correlation between the level of nicotinic acetylcholine receptor (nAChR expression and B lymphocyte differentiation or activation state. Methods. Expression of nAChRs in the REH, Ramos and Daudi cell lines was studied by flow cytometry using nAChR subunit-specific antibodies; cell proliferation was studied by MTT test. Results. It is shown that the level of 42/4 and 7 nAChRs expression increased along with B lymphocyte differentiation (Ramos > REH and activation (Daudi > > Ramos and depended on the antigen-specific receptor expression. The nAChR stimulation/blockade did not influence the intensity of cell proliferation.

Resveratrol (trans-3,5,4'-trihydroxystilbene) suppresses EL4 tumor growth by induction of apoptosis involving reciprocal regulation of SIRT1 and NF-κB.

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Singh, Narendra P; Singh, Udai P; Hegde, Venkatesh L; Guan, Hongbing; Hofseth, Lorne; Nagarkatti, Mitzi; Nagarkatti, Prakash S

2011-08-01

Understanding the molecular mechanisms through which natural products and dietary supplements exhibit anticancer properties is crucial and can lead to drug discovery and chemoprevention. The current study sheds new light on the mode of action of resveratrol (RES), a plant-derived polyphenolic compound, against EL-4 lymphoma growth. Immuno-compromised NOD/SCID mice injected with EL-4 tumor cells and treated with RES (100 mg/kg body weight) showed delayed development and progression of tumor growth and increased mean survival time. RES caused apoptosis in EL4 cells through activation of aryl hydrocarbon receptor (AhR) and upregulation of Fas and FasL expression in vitro. Blocking of RES-induced apoptosis in EL4 cells by FasL mAb, cleavage of caspases and PARP, and release of cytochorme c, demonstrated the participation of both extrinsic and intrinsic pathways of apoptosis. RES also induced upregulation of silent mating type information regulation 2 homolog, 1 (SIRT1) and downregulation of nuclear factor kappa B (NF-κB) in EL4 cells. siRNA-mediated downregulation of SIRT1 in EL4 cells increased the activation of NF-κB but decreased RES-mediated apoptosis, indicating the critical role of SIRT1 in apoptosis via blocking activation of NF-κB. These data suggest that RES-induced SIRT1 upregulation promotes tumor cell apoptosis through negative regulation of NF-κB, leading to suppression of tumor growth. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

B7-H4 as a Target for Breast Cancer Immunotherapy

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2013-06-01

papain proteinase K pronase trypsin B7-H4R B """""" B7-H4R-APC CD25-FITC...Figure 5. B7-H4 receptor is protease resistant. Karpas 299 cells were treated with 0.3 mg/mL papain , 0.025 mg/mL proteinase K, 0.15 mg/mL pronase

A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

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Ricardo Núñez Miguel

2007-08-01

Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

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Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors.

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Lenka Plzakova

Full Text Available Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell-pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria's internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

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Mill, Christopher P.; Gettinger, Kathleen L.; Riese, David J.

2011-01-01

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.

A role of periaqueductal grey NR2B-containing NMDA receptor in mediating persistent inflammatory pain

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Yang Qi

2009-12-01

Full Text Available Abstract The midbrain periaqueductal grey (PAG is a structure known for its roles in pain transmission and modulation. Noxious stimuli potentiate the glutamate synaptic transmission and enhance glutamate NMDA receptor expression in the PAG. However, little is known about roles of NMDA receptor subunits in the PAG in processing the persistent inflammatory pain. The present study was undertaken to investigate NR2A- and NR2B-containing NMDA receptors in the PAG and their modulation to the peripheral painful inflammation. Noxious stimuli induced by hind-paw injection of complete Freund's adjuvant (CFA caused up-regulation of NR2B-containing NMDA receptors in the PAG, while NR2A-containing NMDA receptors were not altered. Whole-cell patch-clamp recordings revealed that NMDA receptor mediated mEPSCs were increased significantly in the PAG synapse during the chronic phases of inflammatory pain in mice. PAG local infusion of Ro 25-6981, an NR2B antagonist, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in rats. Hyperoside (Hyp, one of the flavonoids compound isolated from Rhododendron ponticum L., significantly reversed up-regulation of NR2B-containing NMDA receptors in the PAG and exhibited analgesic activities against persistent inflammatory stimuli in mice. Our findings provide strong evidence that up-regulation of NR2B-containing NMDA receptors in the PAG involves in the modulation to the peripheral persistent inflammatory pain.

GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

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Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco

2008-07-02

Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

Insulin receptor-related receptor as an extracellular pH sensor involved in the regulation of acid-base balance.

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Petrenko, Alexander G; Zozulya, Sergey A; Deyev, Igor E; Eladari, Dominique

2013-10-01

Recent studies of insulin receptor-related receptor (IRR) revealed its unusual property to activate upon extracellular application of mildly alkaline media, pH>7.9. The activation of IRR with hydroxyl anion has typical features of ligand-receptor interaction; it is specific, dose-dependent, involves the IRR extracellular domain and is accompanied by a major conformational change. IRR is a member of the insulin receptor minifamily and has been long viewed as an orphan receptor tyrosine kinase since no peptide or protein agonist of IRR was found. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin-like growth factor receptors in amphibia. The latter two cannot be activated by alkali. Another major difference between them is that unlike ubiquitously expressed insulin and insulin-like growth factor receptors, IRR is found in specific sets of cells of only some tissues, most of them being exposed to extracorporeal liquids of extreme pH. In particular, largest concentrations of IRR are in beta-intercalated cells of the kidneys. The primary physiological function of these cells is to excrete excessive alkali as bicarbonate into urine. When IRR is removed genetically, animals loose the property to excrete bicarbonate upon experimentally induced alkalosis. In this review, we will discuss the available in vitro and in vivo data that support the hypothesis of IRR role as a physiological alkali sensor that regulates acid-base balance. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases. Copyright © 2012 Elsevier B.V. All rights reserved.

cis- and trans-2,3,3a,4,5,9b-Hexahydro-1H-benz[e]indoles: synthesis and evaluation of dopamine D2, and D3 receptor binding affinity

DEFF Research Database (Denmark)

Song, Xiaodong; Crider, Michael A.; Cruse, Sharon F.

1999-01-01

cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz [e]indoles were synthesized as conformationally rigid analogues of 3-phenylpyrrolidine and evaluated for dopamine (DA) D2S and D3 receptor binding affinity. The tricyclic benz[e]indole nucleus was constructed by a previously reported reductive...... configuration. These novel ligands may be useful tools for gaining additional information about the DA D3 receptor. Copyright Elsevier, Paris.dopamine / D2S receptor / D3 receptor / cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz[e]indoles / receptor binding affinity....... receptors was shown by compounds substituted with N-n-propyl or N-allyl groups. The cis-(+-)-N-allyl derivative 21e demonstrated a D2S/D3 selectivity of 290. Resolution of cis-(+-)-5 and trans-(+-)- 21c into individual enantiomers showed that in both series the more active isomer had 3aR absolute...

Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

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Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions.

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Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2014-02-21

We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts*

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Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-01-01

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

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Michihara, Akihiro; Mido, Mayuko; Matsuoka, Hiroshi; Mizutani, Yurika

2015-01-01

A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

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Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

The ROS/NF-κB/NR4A2 pathway is involved in H2O2 induced apoptosis of resident cardiac stem cells via autophagy.

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Shi, Xingxing; Li, Wenjing; Liu, Honghong; Yin, Deling; Zhao, Jing

2017-09-29

Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1 + ) CSCs proved to own therapy potential were cultured and treated with H 2 O 2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H 2 O 2 -induced CSCs apoptosis, thus we demonstrated that H 2 O 2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H 2 O 2 . NR4A2 siRNA attenuated H 2 O 2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF-κB (P65) subunit were both increased by H 2 O 2 . Either the ROS scavenger, N-acetyl-l-cysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H 2 O 2 -induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H 2 O 2 -induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

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Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

2018-05-01

The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

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Xavier Charest-Morin

Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

Receptor protection studies comparing recombinant and native nicotinic receptors: Evidence for a subpopulation of mecamylamine-sensitive native alpha3beta4* nicotinic receptors.

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Free, R Benjamin; Kaser, Daniel J; Boyd, R Thomas; McKay, Dennis B

2006-01-09

Studies involving receptor protection have been used to define the functional involvement of specific receptor subtypes in tissues expressing multiple receptor subtypes. Previous functional studies from our laboratory demonstrate the feasibility of this approach when applied to neuronal tissues expressing multiple nicotinic acetylcholine receptors (nAChRs). In the current studies, the ability of a variety of nAChR agonists and antagonists to protect native and recombinant alpha3beta4 nAChRs from alkylation were investigated using nAChR binding techniques. Alkylation of native alpha3beta4* nAChRs from membrane preparations of bovine adrenal chromaffin cells resulted in a complete loss of specific [(3)H]epibatidine binding. This loss of binding to native nAChRs was preventable by pretreatment with the agonists, carbachol or nicotine. The partial agonist, cytisine, produced partial protection. Several nAChR antagonists were also tested for their ability to protect. Hexamethonium and decamethonium were without protective activity while mecamylamine and tubocurarine were partially effective. Addition protection studies were performed on recombinant alpha3beta4 nAChRs. As with native alpha3beta4* nAChRs, alkylation produced a complete loss of specific [(3)H]epibatidine binding to recombinant alpha3beta4 nAChRs which was preventable by pretreatment with nicotine. However, unlike native alpha3beta4* nAChRs, cytisine and mecamylamine, provide no protection for alkylation. These results highlight the differences between native alpha3beta4* nAChRs and recombinant alpha3beta4 nAChRs and support the use of protection assays to characterize native nAChR subpopulations.

HMGB1 Contributes to the Expression of P-Glycoprotein in Mouse Epileptic Brain through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products.

Directory of Open Access Journals (Sweden)

Yan Chen

Full Text Available The objective of the present study was to investigate the role of high-mobility group box-1 (HMGB1 in the seizure-induced P-glycoprotein (P-gp overexpression and the underlying mechanism. Kainic acid (KA-induced mouse seizure model was used for in vivo experiments. Male C57BL/6 mice were divided into four groups: normal saline control (NS group, KA-induced epileptic seizure (EP group, and EP group pretreated with HMGB1 (EP+HMGB1 group or BoxA (HMGB1 antagonist, EP+BoxA group. Compared to the NS group, increased levels of HMGB1 and P-gp in the brain were observed in the EP group. Injection of HMGB1 before the induction of KA further increased the expression of P-gp while pre-treatment with BoxA abolished this up-regulation. Next, the regulatory role of HMGB1 and its potential involved signal pathways were investigated in mouse microvascular endothelial bEnd.3 cells in vitro. Cells were treated with HMGB1, HMGB1 plus lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS [toll-like receptor 4 (TLR4 antagonist], HMGB1 plus FPS-ZM1 [receptor for advanced glycation end products (RAGE inhibitor], HMGB1 plus SN50 [nuclear factor-kappa B (NF-κB inhibitor], or vehicle. Treatment with HMGB1 increased the expression levels of P-gp, TLR4, RAGE and the activation of NF-κB in bEnd.3 cells. These effects were inhibited by the pre-treatment with either LPS-RS or FPS-ZM1, and were abolished by the pre-treatment of SN50 or a combination treatment of both LPS-RS and FPS-ZM1. Luciferase reporter assays showed that exogenous expression of NF-κB p65 increased the promoter activity of multidrug resistance 1a (P-gp-encoding gene in endothelial cells. These data indicate that HMGB1 contributes to the overexpression of P-gp in mouse epileptic brain tissues via activation of TLR4/RAGE receptors and the downstream transcription factor NF-κB in brain microvascular endothelial cells.

Molecular mechanism for the involvement of nuclear receptor FXR in HBV-associated hepatocellular carcinoma

Directory of Open Access Journals (Sweden)

Yong-dong Niu

2011-08-01

Full Text Available Farnesoid X receptor (FXR, also termed nuclear receptor NR1H4 is critically involved in the regulation of nascent bile formation and bile acid enterohepatic circulation. FXR and bile acids have been shown to play roles in liver regeneration and inflammatory responses. There is increasing evidence suggesting that FXR and the FXR signaling pathway are involved in the pathophysiology of a wide range of liver diseases, such as viral hepatitis, cirrhosis, and hepatocellular carcinoma (HCC. Here we discuss the latest discoveries of FXR functions with relevance to bile acid metabolism and HBV-associated HCC. More specifically, the goal of this review is to discuss the roles of FXR and bile acids in regulating HBV replication and how disregulation of the FXR-bile acid signaling pathway is involved in HBV-associated hepatocarcinogenesis.

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

Science.gov (United States)

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

Science.gov (United States)

Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

2009-12-01

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

Science.gov (United States)

Lv, Can; Mo, Chunheng; Liu, Haikun; Wu, Chao; Li, Zhengyang; Li, Juan; Wang, Yajun

2018-04-20

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary. Copyright © 2018. Published by Elsevier B.V.

Involvement of hippocampal NMDA receptors in retrieval of spontaneous object recognition memory in rats.

Science.gov (United States)

Iwamura, Etsushi; Yamada, Kazuo; Ichitani, Yukio

2016-07-01

The involvement of hippocampal N-methyl-d-aspartate (NMDA) receptors in the retrieval process of spontaneous object recognition memory was investigated. The spontaneous object recognition test consisted of three phases. In the sample phase, rats were exposed to two identical objects several (2-5) times in the arena. After the sample phase, various lengths of delay intervals (24h-6 weeks) were inserted (delay phase). In the test phase in which both the familiar and the novel objects were placed in the arena, rats' novel object exploration behavior under the hippocampal treatment of NMDA receptor antagonist, AP5, or vehicle was observed. With 5 exposure sessions in the sample phase (experiment 1), AP5 treatment in the test phase significantly decreased discrimination ratio when the delay was 3 weeks but not when it was one week. On the other hand, with 2 exposure sessions in the sample phase (experiment 2) in which even vehicle-injected control animals could not discriminate the novel object from the familiar one with a 3 week delay, AP5 treatment significantly decreased discrimination ratio when the delay was one week, but not when it was 24h. Additional experiment (experiment 3) showed that the hippocampal treatment of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, NBQX, decreased discrimination ratio with all delay intervals tested (24h-3 weeks). Results suggest that hippocampal NMDA receptors play an important role in the retrieval of spontaneous object recognition memory especially when the memory trace weakens. Copyright © 2016. Published by Elsevier B.V.

Stereotyped patterns of B-cell receptor in splenic marginal zone lymphoma

KAUST Repository

Zibellini, S.

2010-05-29

Antigen stimulation may be important for splenic marginal zone lymphoma pathogenesis. To address this hypothesis, the occurrence of stereotyped B-cell receptors was investigated in 133 SMZL (26 HCV+) compared with 4,414 HCDR3 sequences from public databases. Sixteen SMZL (12%) showed stereotyped BCR; 7 of 86 (8%) SMZL sequences retrieved from public databases also belonged to stereotyped HCDR3 subsets. Three categories of subsets were identified: i) SMZL-specific subsets (n=5), composed only of 12 SMZL (9 HCV- from our series); ii) Non-Hodgkin\\'s lymphoma-like subsets (n=5), comprising 5 SMZL (4 from our series) clustering with other indolent lymphomas; iii) "CLL-like subsets" (n=6), comprising 6 SMZL (3 from our series) that belonged to known CLL subsets (n=4) or clustered with public CLL sequences. Immunoglobulin 3D modeling of 3 subsets revealed similarities in antigen binding regions not limited to HCDR3. Overall, data suggest that the pathogenesis of splenic marginal zone lymphoma may involve also HCV unrelated epitopes or an antigenic trigger common to other indolent lymphomas. ©2010 Ferrata Storti Foundation.

Stereotyped patterns of B-cell receptor in splenic marginal zone lymphoma

KAUST Repository

Zibellini, S.; Capello, D.; Forconi, F.; Marcatili, P.; Rossi, D.; Rattotti, S.; Franceschetti, S.; Sozzi, E.; Cencini, E.; Marasca, R.; Baldini, L.; Tucci, A.; Bertoni, F.; Passamonti, F.; Orlandi, E.; Varettoni, M.; Merli, M.; Rizzi, S.; Gattei, V.; Tramontano, A.; Paulli, M.; Gaidano, G.; Arcaini, L.

2010-01-01

Antigen stimulation may be important for splenic marginal zone lymphoma pathogenesis. To address this hypothesis, the occurrence of stereotyped B-cell receptors was investigated in 133 SMZL (26 HCV+) compared with 4,414 HCDR3 sequences from public databases. Sixteen SMZL (12%) showed stereotyped BCR; 7 of 86 (8%) SMZL sequences retrieved from public databases also belonged to stereotyped HCDR3 subsets. Three categories of subsets were identified: i) SMZL-specific subsets (n=5), composed only of 12 SMZL (9 HCV- from our series); ii) Non-Hodgkin's lymphoma-like subsets (n=5), comprising 5 SMZL (4 from our series) clustering with other indolent lymphomas; iii) "CLL-like subsets" (n=6), comprising 6 SMZL (3 from our series) that belonged to known CLL subsets (n=4) or clustered with public CLL sequences. Immunoglobulin 3D modeling of 3 subsets revealed similarities in antigen binding regions not limited to HCDR3. Overall, data suggest that the pathogenesis of splenic marginal zone lymphoma may involve also HCV unrelated epitopes or an antigenic trigger common to other indolent lymphomas. ©2010 Ferrata Storti Foundation.

Association of variation in Fcgamma receptor 3B gene copy number with rheumatoid arthritis in Caucasian samples.

NARCIS (Netherlands)

McKinney, C.; Fanciulli, M.; Merriman, M.E.; Phipps-Green, A.; Alizadeh, B.Z.; Koeleman, B.P.; Dalbeth, N.; Gow, P.J.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Riel, P.L.C.M. van; Vyse, T.J.; Aitman, T.J.; Radstake, T.R.D.J.; Merriman, T.R.

2010-01-01

OBJECTIVE: There is increasing evidence that variation in gene copy number (CN) influences clinical phenotype. The low-affinity Fcgamma receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment to sites of inflammation and activation of

Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors

DEFF Research Database (Denmark)

Xochelli, Aliki; Baliakas, Panagiotis; Kavakiotis, Ioannis

2017-01-01

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether...

S100a8/NF-κB signal pathway is involved in the 800-nm diode laser-induced skin collagen remodeling.

Science.gov (United States)

Ren, Xiaolin; Ge, Minggai; Qin, Xiaofeng; Xu, Peng; Zhu, Pingya; Dang, Yongyan; Gu, Jun; Ye, Xiyun

2016-05-01

The 800-nm diode laser is widely used for hair removal and also promotes collagen synthesis, but the molecular mechanism by which dermis responses to the thermal damage induced by the 800-nm diode laser is still unclear. Ten 2-month-old mice were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for PCR, Western blot analysis, and histological study at day 3 or 30 after laser irradiation. The expression of S100a8 and its two receptors (advanced glycosylation end product-specific receptor, RAGE and toll-like receptor 4, TRL4) was upregulated at day 3 after laser treatments. P-p65 levels were also elevated, causing the increase of cytokine (tumor necrosis factor, TNF-α and interleukin 6, IL-6) and MMPs (MMP1a, MMP9). At day 30, PCR and Western blot analysis showed significant increase of type I and III procollagen in the dermis treated with laser. Importantly, skin structure was markedly improved in the laser-irradiated skin compared with the control. Thus, it seemed that S100a8 upregulation triggered NF-κB signal pathway through RAGE and TLR4, responding to laser-induced dermis wound healing. The involvement of the NF-κB pathway in MMP gene transcription promoted the turnover of collagen in the skin, accelerating new collagen synthesis.

Serotonin mediation of early memory formation via 5HT2B receptor-induced glycogenolysis in the day-old chick

Directory of Open Access Journals (Sweden)

Marie Elizabeth Gibbs

2014-04-01

Full Text Available Investigation of the effects of serotonin on memory formation in the chick revealed an action on at least two 5HT receptors. Serotonin injected intracerebrally produced a biphasic effect on memory consolidation with enhancement at low doses and inhibition at higher doses. The non-selective 5HT receptor antagonist methiothepin and the selective 5HT2B/C receptor antagonist SB221284 both inhibited memory, suggesting actions of serotonin on at least 2 different receptor subtypes. The 5HT2B/C and astrocyte-specific 5-HT receptor agonists, fluoxetine and paroxetine, enhanced memory and the effect was attributed to glycogenolysis. Inhibition of glycogenolysis with a low dose of DAB prevented both serotonin and fluoxetine from enhancing memory during short-term memory but not during intermediate memory. The role of serotonin on the 5HT2B/C receptor appears to involve glycogen breakdown in astrocytes during short-term memory, whereas other published evidence attributes the second period of glycogenolysis to noradrenaline.

Effect of vertical sleeve gastrectomy in melanocortin receptor 4-deficient rats

NARCIS (Netherlands)

Mul, J.D.; Begg, D.P.; Alsters, S.I.; van Haaften, G.; Duran, K.J.; D'Alessio, D.A.; le Roux, C.W.; Woods, S.C.; Sandoval, D.A.; Blakemore, A.I.; Cuppen, E.; van Haelst, M.M.; Seeley, R.J.

2012-01-01

Bariatric surgery is currently the most effective treatment for obesity. Vertical sleeve gastrectomy (VSG), a commonly applied bariatric procedure, involves surgically incising most of the volume of the stomach. In humans, partial loss of melanocortin receptor-4 (MC4R) activity is the most common

ErbB receptors and cell polarity: New pathways and paradigms for understanding cell migration and invasion

International Nuclear Information System (INIS)

Feigin, Michael E.; Muthuswamy, Senthil K.

2009-01-01

The ErbB family of receptor tyrosine kinases is involved in initiation and progression of a number of human cancers, and receptor activation or overexpression correlates with poor patient survival. Research over the past two decades has elucidated the molecular mechanisms underlying ErbB-induced tumorigenesis, which has resulted in the development of effective targeted therapies. ErbB-induced signal transduction cascades regulate a wide variety of cell processes, including cell proliferation, apoptosis, cell polarity, migration and invasion. Within tumors, disruption of these core processes, through cooperative oncogenic lesions, results in aggressive, metastatic disease. This review will focus on the ErbB signaling networks that regulate migration and invasion and identify a potential role for cell polarity pathways during cancer progression

Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4

OpenAIRE

Lee, Benhur; Doranz, Benjamin J.; Rana, Shalini; Yi, Yanji; Mellado, Mario; Frade, Jose M. R.; Martinez-A., Carlos; O’Brien, Stephen J.; Dean, Michael; Collman, Ronald G.; Doms, Robert W.

1998-01-01

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor ...

Adenosine A2B Receptors: An Optional Target for the Management of Irritable Bowel Syndrome with Diarrhea?

Directory of Open Access Journals (Sweden)

Teita Asano

2017-11-01

Full Text Available Irritable bowel syndrome (IBS is a functional gastrointestinal disorder, with the characteristic symptoms of chronic abdominal pain and altered bowel habits (diarrhea, constipation, or both. IBS is a highly prevalent condition, which negatively affects quality of life and is a significant burden on global healthcare costs. Although many pharmacological medicines have been proposed to treat IBS, including those targeting receptors, channels, and chemical mediators related to visceral hypersensitivity, successful pharmacotherapy for the disease has not been established. Visceral hypersensitivity plays an important role in IBS pathogenesis. Immune activation is observed in diarrhea-predominant patients with IBS and contributes to the development of visceral hypersensitivity. Adenosine is a chemical mediator that regulates many physiological processes, including inflammation and nociception. Among its receptors, the adenosine A2B receptor regulates intestinal secretion, motor function, and the immune response. We recently demonstrated that the adenosine A2B receptor is involved in visceral hypersensitivity in animal models of IBS. In this review, we discuss the possibility of the adenosine A2B receptor as a novel therapeutic target for IBS.

Complement system proteins which interact with C3b or C4b A superfamily of structurally related proteins

DEFF Research Database (Denmark)

Reid, K B M; Bentley, D R; Campbell, R D

1986-01-01

Recent cDNA sequencing data has allowed the prediction of the entire amino acid sequences of complement components factor B and C2, the complement control proteins factor H and C4b-binding protein and a partial sequence for the Cab/C4b receptor CR1. These proteins all contain internal repeating u...

Reproductive phase dependent daily variation in melatonin receptors (Mel(1a) and Mel(1b)), androgen receptor (AR) and lung associated immunity of Perdicula asiatica.

Science.gov (United States)

Kharwar, R K; Haldar, C

2011-06-01

Our knowledge about the involvement of melatonin in the regulation of lung associated immune system (LAIS) is still poor though the melatonin receptor types (Mel(1a) and Mel(1b)) have been localized in lungs of some wild birds. We thought to explore the correlation between daily variation (within a 24h time scale) in peripheral melatonin and testosterone along with expression of melatonin receptors (Mel(1a) and Mel(1b)) and androgen receptor (AR) in lungs during reproductively active and inactive phases. Receptor expression of Mel(1b) was more prominent than Mel(1a) at all the time points during both the reproductive phases. The expression of AR was inversely related to both the melatonin and its receptor expression at the 24h time scale during both the reproductive phases. Results also reflected a parallel relationship of melatonin, melatonin receptors and all the immune parameters (total leukocyte count, lymphocyte count, % stimulation ratio) suggesting that peripheral melatonin might be responsible for daily periodicity of LAIS. The presence of androgen receptors in lung led us to propose that gonadal steroid does influence the LAIS. Therefore melatonin along with testosterone might be acting as a temporal synchronizer for daily rhythms in lung associated immunity in Perdicula asiatica during different reproductive phases. Copyright © 2011 Elsevier Inc. All rights reserved.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

Science.gov (United States)

Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

Mincle suppresses Toll-like receptor 4 activation.

Science.gov (United States)

Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

2016-07-01

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. © Society for Leukocyte Biology.

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

Energy Technology Data Exchange (ETDEWEB)

Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Germini, Diego; Rodighiero, Isabella [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Mirandola, Prisco [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); De Conto, Flora; Medici, Maria-Cristina [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Gatti, Rita [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); Chezzi, Carlo; Calderaro, Adriana [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy)

2013-05-25

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.

Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

NARCIS (Netherlands)

Lens, S. M.; den Drijver, B. F.; Pötgens, A. J.; Tesselaar, K.; van Oers, M. H.; van Lier, R. A.

1998-01-01

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with

Molecular pathways: the role of NR4A orphan nuclear receptors in cancer.

LENUS (Irish Health Repository)

Mohan, Helen M

2012-06-15

Nuclear receptors are of integral importance in carcinogenesis. Manipulation of classic ligand-activated nuclear receptors, such as estrogen receptor blockade in breast cancer, is an important established cancer therapy. Orphan nuclear receptors, such as nuclear family 4 subgroup A (NR4A) receptors, have no known natural ligand(s). These elusive receptors are increasingly recognized as molecular switches in cell survival and a molecular link between inflammation and cancer. NR4A receptors act as transcription factors, altering expression of downstream genes in apoptosis (Fas-ligand, TRAIL), proliferation, DNA repair, metabolism, cell migration, inflammation (interleukin-8), and angiogenesis (VEGF). NR4A receptors are modulated by multiple cell-signaling pathways, including protein kinase A\\/CREB, NF-κB, phosphoinositide 3-kinase\\/AKT, c-jun-NH(2)-kinase, Wnt, and mitogen-activated protein kinase pathways. NR4A receptor effects are context and tissue specific, influenced by their levels of expression, posttranslational modification, and interaction with other transcription factors (RXR, PPAR-Υ). The subcellular location of NR4A "nuclear receptors" is also important functionally; novel roles have been described in the cytoplasm where NR4A proteins act both indirectly and directly on the mitochondria to promote apoptosis via Bcl-2. NR4A receptors are implicated in a wide variety of malignancies, including breast, lung, colon, bladder, and prostate cancer; glioblastoma multiforme; sarcoma; and acute and\\/or chronic myeloid leukemia. NR4A receptors modulate response to conventional chemotherapy and represent an exciting frontier for chemotherapeutic intervention, as novel agents targeting NR4A receptors have now been developed. This review provides a concise clinical overview of current knowledge of NR4A signaling in cancer and the potential for therapeutic manipulation.

IL-4Rα-associated antigen processing by B cells promotes immunity in Nippostrongylus brasiliensis infection.

Directory of Open Access Journals (Sweden)

William G C Horsnell

2013-10-01

Full Text Available In this study, B cell function in protective T(H2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα⁻/⁻ mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4⁺ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88⁻/⁻ B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4⁺ T cell-mediated protective immunity against N. brasiliensis infection.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

Science.gov (United States)

Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-02-12

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PI3K is involved in PDGF-beta receptor upregulation post-PDGF-BB treatment in mouse HSC.

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Lechuga, Carmen G; Hernández-Nazara, Zamira H; Hernández, Elizabeth; Bustamante, Marcia; Desierto, Gregory; Cotty, Adam; Dharker, Nachiket; Choe, Moran; Rojkind, Marcos

2006-12-01

Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).

DMPD: Signals and receptors involved in recruitment of inflammatory cells. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ow Signals and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and receptors involved in recr...uitment of inflammatory cells. Authors Ben-Baruch A, Mic

Ubiquitylation of an internalized killer cell Ig-like receptor by Triad3A disrupts sustained NF-κB signaling.

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Miah, S M Shahjahan; Purdy, Amanda K; Rodin, Nicholas B; MacFarlane, Alexander W; Oshinsky, Jennifer; Alvarez-Arias, Diana A; Campbell, Kerry S

2011-03-01

Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.

Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4

NARCIS (Netherlands)

Gascan, H.; Aversa, G. G.; Gauchat, J. F.; van Vlasselaer, P.; Roncarolo, M. G.; Yssel, H.; Kehry, M.; Spits, H.; de Vries, J. E.

1992-01-01

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell

Ovis aries CR4 is involved in Mannheimia haemolytica leukotoxin-induced cytotoxicity.

Science.gov (United States)

Lawrence, Paulraj K; Dassanayake, Rohana P

2010-06-15

Pneumonia caused by Mannheimia haemolytica is an important disease of domestic sheep (DS, Ovis aries) and cattle (BO). M. haemolytica is a normal commensal of the upper respiratory tract in ruminants, but during stress and viral infection it breaches the host innate mucosal defense and descents into lungs causing fibrinous pleuropneumonia. Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all subsets of ruminant leukocytes. Earlier, we and others have shown that DS and BO LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) can mediate Lkt-induced cytolysis. It is not clear whether CR4 (CD11c/CD18), which is involved in chemotaxis, phagocytosis and regulates host immune response can also mediate Lkt-induced cytolysis in ruminants. The host innate immune response to M. haemolytica is poorly understood and the involvement of CR4 in M. haemolytica pathogenesis is one of the most understudied. This problem is further compounded by the lack of cd11c genes from any ruminant species. Therefore, the objectives of this study were to clone cd11c and determine whether CR4 can serve as a receptor for Lkt. In this direction we cloned two alleles of cd11c gene from leukocytes isolated from DS blood by RT-PCR. Transfectants developed expressing functional DS CR4 were found to be cytotoxic to Lkt from four different isolates of M. haemolytica. This is the first report confirming the ability of a recombinant ovine CR4 to bind to M. haemolytica Lkt and mediate concentration-dependent lysis of host cells, thus, confirming their role in M. haemolytica pathogenesis. This is a critical step in understanding host innate immunity and the management of pneumonia in sheep. Copyright 2009 Elsevier B.V. All rights reserved.

Immune receptors involved in Streptococcus suis recognition by dendritic cells.

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Marie-Pier Lecours

Full Text Available Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. The objective of this study was to identify the immune receptors involved in S. suis recognition by dendritic cells (DCs. Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR signaling. Supporting this fact, TLR2(-/- DCs were severely impaired in the release of several cytokines and the surface expression of CD86 and MHC-II. The release of IL-12p70 and CXC10, and the expression of CD40 were found to depend on signaling by both TLR2 and TLR9. The release of IL-23 and CXCL1 were partially dependent on NOD2. Finally, despite the fact that MyD88 signaling was crucial for DC activation and maturation, MyD88-dependent pathways were not implicated in S. suis internalization by DCs. This first study on receptors involved in DC activation by S. suis suggests a major involvement of MyD88 signaling pathways, mainly (but not exclusively through TLR2. A multimodal recognition involving a combination of different receptors seems essential for DC effective response to S. suis.

Human eosinophils - potential pharmacological model applied in human histamine H4 receptor research.

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Grosicki, Marek; Kieć-Kononowicz, Katarzyna

2015-01-01

Histamine and histamine receptors are well known for their immunomodulatory role in inflammation. In this review we describe the role of histamine and histamine H4 receptor on human eosinophils. In the first part of article we provide short summary of histamine and histamine receptors role in physiology and histamine related therapeutics used in clinics. We briefly describe the human histamine receptor H4 and its ligands, as well as human eosinophils. In the second part of the review we provide detailed description of known histamine effects on eosinophils including: intracellular calcium concentration flux, actin polymerization, cellular shape change, upregulation of adhesion proteins and cellular chemotaxis. We provide proofs that these effects are mainly connected with the activation of histamine H4 receptor. When examining experimental data we discuss the controversial results and limitations of the studies performed on isolated eosinophils. In conclusion we believe that studies on histamine H4 receptor on human eosinophils can provide interesting new biomarkers that can be used in clinical studies of histamine receptors, that in future might result in the development of new strategies in the treatment of chronic inflammatory conditions like asthma or allergy, in which eosinophils are involved.

Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis.

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Kahn, Robin; Mossberg, Maria; Ståhl, Anne-Lie; Johansson, Karl; Lopatko Lindman, Ingrid; Heijl, Caroline; Segelmark, Mårten; Mörgelin, Matthias; Leeb-Lundberg, L M Fredrik; Karpman, Diana

2017-01-01

During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation. By flow cytometry, plasma from patients with vasculitis were found to contain high levels of leukocyte-derived microvesicles bearing B1-receptors. Importantly, renal biopsies from two patients with vasculitis showed leukocyte-derived microvesicles bearing B1-receptors docking on glomerular endothelial cells providing in vivo relevance. Microvesicles derived from B1-receptor-transfected human embryonic kidney cells transferred B1-receptors to wild-type human embryonic kidney cells, lacking the receptor, and to glomerular endothelial cells. The transferred B1-receptors induced calcium influx after B1-receptor agonist stimulation: a response abrogated by a specific B1-receptor antagonist. Microvesicles derived from neutrophils also transferred B1-receptors to wild-type human embryonic kidney cells and induced calcium influx after stimulation. Thus, we found a novel mechanism by which microvesicles transfer functional receptors and promote kinin-associated inflammation. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Association of variation in Fc gamma receptor 3B gene copy number with rheumatoid arthritis in Caucasian samples

NARCIS (Netherlands)

McKinney, Cushla; Fanciulli, Manuela; Merriman, Marilyn E.; Phipps-Green, Amanda; Alizadeh, Behrooz Z.; Koeleman, Bobby P. C.; Dalbeth, Nicola; Gow, Peter J.; Harrison, Andrew A.; Highton, John; Jones, Peter B.; Stamp, Lisa K.; Steer, Sophia; Barrera, Pilar; Coenen, Marieke J. H.; Franke, Barbara; van Riel, Piet L. C. M.; Vyse, Tim J.; Aitman, Tim J.; Radstake, Timothy R. D. J.; Merriman, Tony R.

2010-01-01

Objective There is increasing evidence that variation in gene copy number (CN) influences clinical phenotype. The low-affinity Fc gamma receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment to sites of inflammation and activation of

Leukotriene B4—leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8+ T cells

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Lv, Jiaoyan; Zou, Linlin; Zhao, Lina; Yang, Wei; Xiong, Yingluo; Li, Bingji; He, Rui

2015-01-01

Leukotriene B4 (LTB4) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8+ T cells. The role of the LTB4–BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8+ T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8+ T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8+ T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4–BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8+ T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis. PMID:25959240

Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

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Francisco J Quintana

Full Text Available B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

Kinin B1 receptors mediate depression-like behavior response in stressed mice treated with systemic E. coli lipopolysaccharide

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Campos Maria M

2010-12-01

Full Text Available Abstract Background Kinin B1 receptors are inducible molecules up-regulated after inflammatory stimuli. This study evaluated the relevance of kinin B1 receptors in a mouse depression behavior model. Methods Mice were exposed to a 5-min swimming session, and 30 min later they were injected with E. coli lipopolysaccharide (LPS. Depression-like behavior was assessed by determining immobility time in a tail suspension test. Different brain structures were collected for molecular and immunohistochemical studies. Anhedonia was assessed by means of a sucrose intake test. Results Our protocol elicited an increase in depression-like behavior in CF1 mice, as assessed by the tail-suspension test, at 24 h. This behavior was significantly reduced by treatment with the selective B1 receptor antagonists R-715 and SSR240612. Administration of SSR240612 also prevented an increase in number of activated microglial cells in mouse hippocampus, but did not affect a reduction in expression of mRNA for brain-derived neurotrophic factor. The increased immobility time following LPS treatment was preceded by an enhancement of hippocampal and cortical B1 receptor mRNA expression (which were maximal at 1 h, and a marked production of TNFα in serum, brain and cerebrospinal fluid (between 1 and 6 h. The depression-like behavior was virtually abolished in TNFα p55 receptor-knockout mice, and increased B1 receptor mRNA expression was completely absent in this mouse strain. Furthermore, treatment with SSR240612 was also effective in preventing anhedonia in LPS-treated mice, as assessed using a sucrose preference test. Conclusion Our data show, for the first time, involvement of kinin B1 receptors in depressive behavioral responses, in a process likely associated with microglial activation and TNFα production. Thus, selective and orally active B1 receptor antagonists might well represent promising pharmacological tools for depression therapy.

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A2B adenosine receptors.

Science.gov (United States)

Antonioli, Luca; Pellegrini, Carolina; Fornai, Matteo; Tirotta, Erika; Gentile, Daniela; Benvenuti, Laura; Giron, Maria Cecilia; Caputi, Valentina; Marsilio, Ilaria; Orso, Genny; Bernardini, Nunzia; Segnani, Cristina; Ippolito, Chiara; Csóka, Balázs; Németh, Zoltán H; Haskó, György; Scarpignato, Carmelo; Blandizzi, Corrado; Colucci, Rocchina

2017-12-01

Adenosine A 2B receptors (A 2B R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A 2B R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A 2B R localization was examined by immunofluorescence. The role of A 2B R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A 2B R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A 2B R antagonist MRS1754 enhanced electrically induced NK 1 -mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A 2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A 2B R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A 2B R expression. A 2B R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

Effects of a novel bradykinin B1 receptor antagonist and angiotensin II receptor blockade on experimental myocardial infarction in rats.

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Dongmei Wu

Full Text Available The aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1 receptor antagonist after MI in rats.Sprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823 or AT1 receptor antagonist (irbesartan alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP; greater first derivative of left ventricular pressure (± dp/dt max, left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2 and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1 mRNA expression were not significantly affected by B1 receptor blockade.The present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI.

Effect of increased HoxB4 on human megakaryocytic development

International Nuclear Information System (INIS)

Zhong, Yiming; Sullenbarger, Brent; Lasky, Larry C.

2010-01-01

Research highlights: → HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. → HoxB4 fusion protein enhanced megakaryocytic development of CD34 + cord blood cells. → Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. → HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord

Effect of increased HoxB4 on human megakaryocytic development

Energy Technology Data Exchange (ETDEWEB)

Zhong, Yiming [Department of Pathology, The Ohio State University, Columbus, OH (United States); Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH (United States); Sullenbarger, Brent [Department of Pathology, The Ohio State University, Columbus, OH (United States); Lasky, Larry C., E-mail: Lasky.4@osu.edu [Department of Pathology, The Ohio State University, Columbus, OH (United States); Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH (United States)

2010-07-30

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34

Suppressing effect of COR659 on alcohol, sucrose, and chocolate self-administration in rats: involvement of the GABAB and cannabinoid CB1 receptors.

Science.gov (United States)

Maccioni, Paola; Colombo, Giancarlo; Lorrai, Irene; Zaru, Alessandro; Carai, Mauro A M; Gessa, Gian Luigi; Brizzi, Antonella; Mugnaini, Claudia; Corelli, Federico

2017-09-01

COR659 [methyl2-(4-chlorophenylcarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate] is a new, positive allosteric modulator (PAM) of the GABA B receptor. This study evaluated whether COR659 shared with previously tested GABA B PAMs the capacity to reduce alcohol self-administration in rats. Treatment with non-sedative doses of COR659 (2.5, 5, and 10 mg/kg; i.p.) suppressed lever-responding for alcohol (15% v/v) in Sardinian alcohol-preferring (sP) rats under the fixed ratio (FR) 4 (FR4) and progressive ratio (PR) schedules of reinforcement; COR659 was more potent and effective than the reference GABA B PAM, GS39783. Treatment with COR659, but not GS39783, suppressed (a) lever-responding for a sucrose solution (1-3% w/v) in sP rats under the FR4 and PR schedules, (b) lever-responding for a chocolate solution [5% (w/v) Nesquik®] in Wistar rats under the FR10 and PR schedules, and (c) cue-induced reinstatement of chocolate seeking in Wistar rats. Treatment with COR659 was completely ineffective on lever-responding (FR10) for regular food pellets in food-deprived Wistar rats. Pretreatment with the GABA B receptor antagonist, SCH50911, partially blocked COR659-induced reduction of alcohol self-administration, being ineffective on reduction of chocolate self-administration. Pretreatment with the cannabinoid CB 1 receptor antagonist, AM4113, fully blocked COR659-induced reduction of chocolate self-administration, being ineffective on reduction of alcohol self-administration. COR659 might exert its behavioral effects via a composite mechanism: (i) positive allosteric modulation of the GABA B receptor, responsible for a large proportion of reduction of alcohol self-administration; (ii) an action at other receptor system(s), including the cannabinoid CB 1 receptor, through which COR659 affects seeking and consumption of highly palatable foods.

Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces

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Brown Scott

2010-07-01

Full Text Available Abstract Background Interleukin-4 (IL4 is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths 1. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution. Results This analysis revealed evidence of diversifying selection at 15 residues, clustered in epitopes responsible for IL4 binding to its Type I and Type II receptors. Such a striking signature of selective pressure suggested either recurrent episodes of pathogen antagonism or ligand/receptor co-evolution. To test the latter possibility, we performed detailed functional analysis of IL4 allotypes expressed by Mus musculus musculus and Mus musculus castaneus, which happen to differ at 5 residues (including three at positively selected sites in and adjacent to the site 1 epitope that binds the IL4Rα subunit shared by the Type I and Type II IL4 receptors. We show that this intra-species variation affects the ability of IL4 neither to bind IL4 receptor alpha (IL4Rα nor to signal biological responses through its Type I receptor. Conclusions Our results -- reminiscent of clustered positively selected sites revealing functionally important residues at host-virus interaction interfaces -- are consistent with IL4 having evolved to avoid recurrent pathogen antagonism, while maintaining the capacity to bind and signal through its cognate receptor. This work exposes what may be a general feature of evolutionary conflicts fought by pathogen antagonists at host protein-protein interaction interfaces involved in immune signaling: the emergence of receptor-binding ligand epitopes capable of buffering amino acid variation.

Obesity is associated with high serotonin 4 receptor availability in the brain reward circuitry

DEFF Research Database (Denmark)

Haahr, M. E.; Rasmussen, Peter Mondrup; Madsen, K.

2012-01-01

in food intake, and that pharmacological or genetic manipulation of the receptor in reward-related brain areas alters food intake.Here, we used positron emission tomography in humans to examine the association between cerebral 5-HT4Rs and common obesity.We found in humans a strong positive association......The neurobiology underlying obesity is not fully understood. The neurotransmitter serotonin (5-HT) is established as a satiety-generating signal, but its rewarding role in feeding is less well elucidated. From animal experiments there is now evidence that the 5-HT4 receptor (5-HT4R) is involved......'s food intake. They also suggest that pharmacological stimulation of the cerebral 5-HT4R may reduce reward-related overeating in humans....

Identification and functional analysis of pheromone and receptor genes in the B3 mating locus of Pleurotus eryngii.

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Kim, Kyung-Hee; Kang, Young Min; Im, Chak Han; Ali, Asjad; Kim, Sun Young; Je, Hee-Jeong; Kim, Min-Keun; Rho, Hyun Su; Lee, Hyun Sook; Kong, Won-Sik; Ryu, Jae-San

2014-01-01

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

Kinetic modeling of 11C-SB207145 binding to 5-HT4 receptors in the human brain in vivo

DEFF Research Database (Denmark)

Marner, Lisbeth; Gillings, Nic; Comley, Robert A

2009-01-01

The serotonin 4 receptor (5-HT(4) receptor) is known to be involved in learning and memory. We evaluated for the first time the quantification of a novel 5-HT(4) receptor radioligand, (11)C-SB207145, for in vivo brain imaging with PET in humans. METHODS: For evaluation of reproducibility, 6...

Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

Science.gov (United States)

Miller, Eileen; Czopek, Alicja; Duthie, Karolina M; Kirkby, Nicholas S; van de Putte, Elisabeth E Fransen; Christen, Sibylle; Kimmitt, Robert A; Moorhouse, Rebecca; Castellan, Raphael F P; Kotelevtsev, Yuri V; Kuc, Rhoda E; Davenport, Anthony P; Dhaun, Neeraj; Webb, David J; Hadoke, Patrick W F

2017-02-01

The role of smooth muscle endothelin B (ET B ) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ET B receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ET B receptors were selectively deleted from smooth muscle by crossing floxed ET B mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ET B deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ET B was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ET B -mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ET B -mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ET B knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ET B blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ET B -mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ET B knockout mice. In the absence of other pathology, ET B receptors in vascular smooth muscle make a small but significant contribution to ET B -dependent regulation of BP. These ET B receptors have no effect on vascular contraction or neointimal remodeling. © 2016 The Authors.

Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

Science.gov (United States)

Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

2006-01-01

An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

Androgen Receptor Involvement in Rat Amelogenesis: An Additional Way for Endocrine-Disrupting Chemicals to Affect Enamel Synthesis.

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Jedeon, Katia; Loiodice, Sophia; Salhi, Khaled; Le Normand, Manon; Houari, Sophia; Chaloyard, Jessica; Berdal, Ariane; Babajko, Sylvie

2016-11-01

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERβ remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

An allosteric enhancer of M4muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

DEFF Research Database (Denmark)

Dencker, Ditte; Weikop, Pia; Sørensen, Gunnar

2012-01-01

The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M4 acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M4 receptors could...

The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

Energy Technology Data Exchange (ETDEWEB)

Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

2015-11-30

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

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Alena Z Minton

Full Text Available Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1 is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B receptors in the retina, mainly in retinal ganglion cells (RGCs, nerve fiber layer (NFL, and also in the inner plexiform layer (IPL and inner nuclear layer (INL. To determine the role of ET(B receptors in neurodegeneration, Wistar-Kyoto wild type (WT and ET(B receptor-deficient (KO rats were subjected to retrograde labeling with Fluoro-Gold (FG, following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

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Dániel Szili

Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

Differential involvement of cortical muscarinic and NMDA receptors in short- and long-term taste aversion memory.

Science.gov (United States)

Ferreira, G; Gutiérrez, R; De La Cruz, V; Bermúdez-Rattoni, F

2002-09-01

In conditioned taste aversion, an animal avoids a taste previously associated with toxic effects, and this aversive memory formation requires an intact insular cortex. In this paper, we investigated the possible differential involvement of cholinergic and glutamatergic receptors in the insular cortex in short-term memory (STM) and long-term memory (LTM) of taste aversion in rats. Taste aversion was induced by intraperitoneal administration of lithium chloride (a malaise-inducing drug) 15 min after experience with an unfamiliar taste. In order to test STM and LTM of taste aversion, taste stimulus was again presented 4 h and 72 h after lithium injection, respectively. During the acquisition, microinjection of the muscarinic antagonist, scopolamine, in the insular cortex before, but not after, the presentation of the new taste, abolished STM as well as LTM. Blockade of the NMDA receptor, in the insular cortex, by AP5 before, but not after, the presentation of the taste stimulus, impaired LTM but left STM intact. Moreover, when injected 1 h after malaise induction (i.e., during taste-illness association), AP5 disrupted both STM and LTM. These results suggest that activation of muscarinic receptors in the insular cortex is involved in the acquisition of taste memory, whereas NMDA receptors participate in taste memory consolidation. These data demonstrate that different neurochemical mechanisms subserve different memory phases. NMDA receptors are also probably involved in processing the visceral input, thus allowing subsequent taste-illness association. This indicates that in the same cortical area the same neurotransmitter system can be involved in distinct processes: taste memory consolidation vs. taste-illness association.

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

Science.gov (United States)

Joseph, Lauren; Thomsen, Morgane

2017-06-30

Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth.

Science.gov (United States)

Xue, Kai; Liu, Jia-yin; Murphy, Bruce D; Tsang, Benjamin K

2012-12-01

Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5α-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[11C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

International Nuclear Information System (INIS)

Christiaans, Johannes A.M.; Klein, Pieter J.; Metaxas, Athanasios; Kooijman, Esther J.M.; Schuit, Robert C.; Leysen, Josée E.; Lammertsma, Adriaan A.; Berckel, Bart N.M. van; Windhorst, Albert D.

2014-01-01

Introduction: The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. Methods: N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. Results: N-((5-(4-fluoro-2-[ 11 C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49 ± 3% (decay-corrected) yield, affording 4.1 ± 0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of > 99% and with a specific activity of 78 ± 10 GBq/μmol. Conclusion: A new NR2B PET ligand was developed in high yield. [ 11 C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [ 11 C]4 as an NR2B PET ligand

Toll-like receptor 4-positive macrophages protect mice from Pasteurella pneumotropica-induced pneumonia

Science.gov (United States)

Hart, Marcia L.; Mosier, Derek A.; Chapes, Stephen K.

2003-01-01

This study investigates Toll-like receptor 4 (TLR4)-positive macrophages in early recognition and clearance of pulmonary bacteria. TLR4 is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide of gram-negative bacteria. The TLR4(Lps-del) mouse strains C57BL10/ScN (B10) and STOCK Abb(tm1) TLR4(Lps-del) Slc11a1(s)(B10 x C2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these mice have the TLR4(Lps-del) genotype, we hypothesized that reconstitution of mice with TLR4-positive macrophages would provide resistance to this bacterium. A cultured macrophage cell line (C2D macrophages) and bone marrow cells from C2D mice were adoptively transferred to B10 and B10 x C2D mice by intraperitoneal injection. C2D macrophages increased B10 and B10 x C2D mouse resistance to P. pneumotropica. In C2D-recipient mice there was earlier transcription of tumor necrosis factor alpha and chemokines JE and macrophage inflammatory protein 2 (MIP-2) in the lungs of B10 and B10 x C2D mice, and there was earlier transcription of KC and MIP-1alpha in B10 x C2D mice. In addition, the course of inflammation following experimental Pasteurella challenge was altered in C2D recipients. C2D macrophages also protected B10 x C2D mice, which lack CD4(+) T cells. These data indicate that macrophages are critical for pulmonary immunity and can provide host resistance to P. pneumotropica. This study indicates that TLR4-positive macrophages are important for early recognition and clearance of pulmonary bacterial infections.

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

Science.gov (United States)

Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

2018-06-09

Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

The anticonvulsant gabapentin (neurontin) does not act through gamma-aminobutyric acid-B receptors

DEFF Research Database (Denmark)

Jensen, Anders A.; Mosbacher, Johannes; Elg, Susanne

2002-01-01

The actions of the anticonvulsant gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] have been somewhat enigmatic until recently, when it was claimed to be a gamma-aminobutyric acid-B (GABA(B)) receptor agonist acting exclusively at a heterodimeric complex containing the GABA(B(1a...... in vitro assays. In light of these results, we find it highly questionable that gabapentin is a GABA(B) receptor agonist. Hence, the anticonvulsive effects of the compound have to arise from GABA(B) receptor-independent mechanisms. This also implies that the first GABA(B) receptor splice variant...

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch.

Science.gov (United States)

Jian, Tunyu; Yang, Niuniu; Yang, Yan; Zhu, Chan; Yuan, Xiaolin; Yu, Guang; Wang, Changming; Wang, Zhongli; Shi, Hao; Tang, Min; He, Qian; Lan, Lei; Wu, Guanyi; Tang, Zongxiang

2016-01-01

Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG) neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip)-a histamine H4 receptor special agonist under cutaneous injection-obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3-50 μM) could also induce a dose-dependent increase in intracellular Ca(2+) ([Ca(2+)]i) of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca(2+) responses. In addition, immepip-induced [Ca(2+)]i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons' responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation.

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch

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Tunyu Jian

2016-01-01

Full Text Available Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip—a histamine H4 receptor special agonist under cutaneous injection—obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3–50 μM could also induce a dose-dependent increase in intracellular Ca2+ (Ca2+i of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca2+ responses. In addition, immepip-induced Ca2+i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons’ responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation.

Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ9-tetrahydrocannabinol in human CD4+ T cells

International Nuclear Information System (INIS)

Ngaotepprutaram, Thitirat; Kaplan, Barbara L.F.; Kaminski, Norbert E.

2013-01-01

We have previously reported that Δ 9 -tetrahydrocannabinol (Δ 9 -THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4 + T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ 9 -THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ 9 -THC attenuated CD40L expression in human CD4 + T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ 9 -THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ 9 -THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ 9 -THC suppresses human T cell function. - Highlights: • Δ 9 -THC attenuated CD40L expression in activated human CD4+ T cells. • Δ 9 -THC suppressed DNA-binding activity of NFAT and NFκB. • Δ 9 -THC impaired elevation of intracellular Ca2+. • Δ 9 -THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

International Nuclear Information System (INIS)

Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

2006-01-01

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

Genetic interactions between neurofibromin and endothelin receptor B in mice.

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Mugdha Deo

Full Text Available When mutations in two different genes produce the same mutant phenotype, it suggests that the encoded proteins either interact with each other, or act in parallel to fulfill a similar purpose. Haploinsufficiency of Neurofibromin and over-expression of Endothelin 3 both cause increased numbers of melanocytes to populate the dermis during mouse development, and thus we are interested in how these two signaling pathways might intersect. Neurofibromin is mutated in the human genetic disease, neurofibromatosis type 1, which is characterized by the development of Schwann cell based tumors and skin hyper-pigmentation. Neurofibromin is a GTPase activating protein, while the Endothelin 3 ligand activates Endothelin receptor B, a G protein coupled receptor. In order to study the genetic interactions between endothelin and neurofibromin, we defined the deletion breakpoints of the classical Ednrb piebald lethal allele (Ednrb(s-l and crossed these mice to mice with a loss-of-function mutation in neurofibromin, Dark skin 9 (Dsk9. We found that Neurofibromin haploinsufficiency requires Endothelin receptor B to darken the tail dermis. In contrast, Neurofibromin haploinsufficiency increases the area of the coat that is pigmented in Endothelin receptor B null mice. We also found an oncogenic mutation in the G protein alpha subunit, GNAQ, which couples to Endothelin receptor B, in a uveal melanoma from a patient with neurofibromatosis type 1. Thus, this data suggests that there is a complex relationship between Neurofibromin and Endothelin receptor B.

Clinical Significance of ErbB Receptor Family in Urothelial Carcinoma of the Bladder: A Systematic Review and Meta-Analysis

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Yuh-Shyan Tsai

2012-01-01

Full Text Available The prognostic importance of examining ErbB receptor family expression in human bladder cancer remains uncertain. Using published evidence, we examined the clinical value and the updated results of clinical trials targeting ErbB receptor family members. Twenty-seven articles from 65 references related to ErbB receptor expression assessment in bladder cancer were reviewed. The estimates included the association significance, hazard ratios, and 95% confidence intervals (CIs from actuarial curves and survival analyses. A meta-analysis was done on those reports using univariate log-rank tests or a Cox-regression model. The methods of analysis and study subjects chosen varied widely among studies. The overall risks of disease progression for patients with EGFR or ErbB2 overexpression were 4.5 (95% CI: 2.5–8.4 and 1.1 (95% CI: 0.6–1.9, and the risks of mortality were 3.0 (95% CI: 1.6–5.9 and 1.1 (95% CI: 1.0–1.2, respectively. However, the significance of coexpression patterns of the ErbB receptor family remains controversial. None of six clinical trials yielded convincing results for blockading ErbB receptor signaling in urothelial carcinoma. The results of this analysis suggest that assessing co-expression patterns of the ErbB family may provide better prognostic information for bladder cancer patients.

Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF-κB and TGF-β1/Smad2 Signaling Pathways

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Liteng Lin

2018-01-01

Full Text Available Liver fibrosis is characterized by activated hepatic stellate cells (HSC and extracellular matrix accumulation. Blocking the activation of HSC and the inflammation response are two major effective therapeutic strategies for liver fibrosis. In addition to the long history of using andrographolide (Andro for inflammatory disorders, we aimed at elucidating the pharmacological effects and potential mechanism of Andro on liver fibrosis. In this study, liver fibrosis was induced by carbon tetrachloride (CCl4 and the mice were intraperitoneally injected with Andro for 6 weeks. HSC cell line (LX-2 and primary HSC were also treated with Andro in vitro. Treatment of CCl4-induced mice with Andro decreased the levels of alanine aminotransferase (ALT and aspartate aminotransferase (AST, Sirius red staining as well as the expression of α smooth muscle actin (α-SMA and transforming growth factor- (TGF- β1. Furthermore, the expression of Toll-like receptor (TLR4 and NF-κB p50 was also inhibited by Andro. Additionally, in vitro data confirmed that Andro treatment not only attenuated the expression of profibrotic and proinflammatory factors but also blocked the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways. These results demonstrate that Andro prevents liver inflammation and fibrosis, which is in correlation with the inhibition of the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways, highlighting Andro as a potential therapeutic strategy for liver fibrosis.

Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice.

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Lee, Min Jung; Cho, Kang Hun; Park, Hyun Min; Sung, Hyun Jung; Choi, Sunghak; Im, Weonbin

2014-07-15

DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation. Copyright © 2014 Elsevier B.V. All rights reserved.

Gamma-hydroxybutyric acid (GHB) and the mesoaccumbens reward circuit: evidence for GABA(B) receptor-mediated effects.

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Pistis, M; Muntoni, A L; Pillolla, G; Perra, S; Cignarella, G; Melis, M; Gessa, G L

2005-01-01

Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid naturally occurring in the mammalian brain, which recently emerged as a major recreational drug of abuse. GHB has multiple neuronal mechanisms including activation of both the GABA(B) receptor, and a distinct GHB-specific receptor. This complex GHB-GABA(B) receptor interaction is probably responsible for the multifaceted pharmacological, behavioral and toxicological profile of GHB. Drugs of abuse exert remarkably similar effects upon reward-related circuits, in particular the mesolimbic dopaminergic system and the nucleus accumbens (NAc). We used single unit recordings in vivo from urethane-anesthetized rats to characterize the effects of GHB on evoked firing in NAc "shell" neurons and on spontaneous activity of antidromically identified dopamine (DA) cells located in the ventral tegmental area. GHB was studied in comparison with the GABA(B) receptor agonist baclofen and antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH50911). Additionally, we utilized a GHB analog, gamma-(p-methoxybenzil)-gamma-hydroxybutyric acid (NCS-435), devoid of GABA(B) binding properties, but with high affinity for specific GHB binding sites. In common with other drugs of abuse, GHB depressed firing in NAc neurons evoked by the stimulation of the basolateral amygdala. On DA neurons, GHB exerted heterogeneous effects, which were correlated to the baseline firing rate of the cells but led to a moderate stimulation of the DA system. All GHB actions were mediated by GABA(B) receptors, since they were blocked by SCH50911 and were not mimicked by NCS-435. Our study indicates that the electrophysiological profile of GHB is close to typical drugs of abuse: both inhibition of NAc neurons and moderate to strong stimulation of DA transmission are distinctive features of diverse classes of abused drugs. Moreover, it is concluded that addictive and rewarding properties of GHB do not necessarily involve a putative high affinity GHB

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

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Cameron, W.; Doyle, K.; Rocklin, R.E.

1986-01-01

A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

Estradiol-induced increase in the magnitude of long-term potentiation is prevented by blocking NR2B-containing receptors.

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Smith, Caroline C; McMahon, Lori L

2006-08-16

Estradiol, through activation of genomic estrogen receptors, induces changes in synaptic morphology and function in hippocampus, a brain region important for memory acquisition. Specifically, this hormone increases CA1 pyramidal cell dendritic spine density, NMDA receptor (NMDAR)-mediated transmission, and the magnitude of long-term potentiation (LTP) at CA3-CA1 synapses. We recently reported that the estradiol-induced increase in LTP magnitude occurs only when there is a simultaneous increase in the fractional contribution of NMDAR-mediated transmission relative to AMPA receptor transmission, suggesting a direct role for the increase in NMDAR transmission to the heightened LTP magnitude. Estradiol has been shown to increase expression of the NMDAR subunit NR2B, but whether this translates into an increase in function of NR2B-containing receptors remains to be determined. Here we show that not only is the estradiol-induced increase in NMDAR transmission mediated by NR2B-containing receptors, but blocking these receptors using RO25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol] (0.5 microM), an NR2B selective antagonist, prevents the estradiol-induced increase in LTP magnitude. Thus, our data show a causal link between the estradiol-induced increase in transmission mediated by NR2B-containing NMDARs and the increase in LTP magnitude.

Design and Synthesis of a Series of L-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine

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Krogsgaard-Larsen, Niels; Delgar, Claudia; Koch, Karina

2017-01-01

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxy-phenoxy)pyrrolidine-2-carboxylic acid (1b...... to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C,O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents......), for cloned homomeric kainic acid receptor subtype 1 (GluK1) was attained (Ki = 4 µM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 µM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared...

Familial Risk for Major Depression is Associated with Lower Striatal 5-HT4 Receptor Binding

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Madsen, Karine; Torstensen, Eva; Holst, Klaus K

2014-01-01

was to determine whether familial risk for MDD is associated with cerebral 5-HT4 receptor binding as measured with [(11)C]SB207145 brain PET imaging. Familial risk is the most potent risk factor of MDD. METHODS: We studied 57 healthy individuals (mean age 36 yrs, range 20-86; 21 women), 26 of which had first......-degree relatives treated for MDD. RESULTS: We found that having a family history of MDD was associated with lower striatal 5-HT4 receptor binding (p = 0.038; in individuals below 40 years, p = 0.013). Further, we found evidence for a "risk-dose effect" on 5-HT4 receptor binding, since the number of first......-degree relatives with a history of MDD binding correlated negatively with 5-HT4 receptor binding in both the striatum (p = 0.001) and limbic regions (p = 0.012). CONCLUSIONS: Our data suggest that the 5-HT4 receptor is involved in the neurobiological mechanism underlying familial risk for depression...

Diclofenac pretreatment effects on the toll-like receptor 4/nuclear factor kappa B-mediated inflammatory response to eccentric exercise in rat liver.

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Barcelos, Rômulo Pillon; Bresciani, Guilherme; Rodriguez-Miguelez, Paula; Cuevas, Maria José; Soares, Félix Alexandre Antunes; Barbosa, Nilda Vargas; González-Gallego, Javier

2016-03-01

Acute exercise is a stress stimulus that may cause cell damage through the activation of the toll-like receptor (TLR)4 pathway, resulting in the translocation of nuclear factor kappa B (NF-κB) into the cell nucleus and the upregulation of inflammatory genes. Nonsteroidal anti-inflammatory drugs, such as diclofenac, are often prescribed to counteract exercise-induced inflammation. This study analyzed effects of diclofenac pretreatment on the TLR4/NF-κB pathway in rat liver after an acute eccentric exercise. Twenty male Wistar rats were divided in four groups: control-saline, control-diclofenac, exercise-saline and exercise-diclofenac. The rats received saline or diclofenac (10mg/kg) for 7days prior to an eccentric exercise bout. After exercise there was an increase in TLR4, myeloid differentiation primary response gene 88 (MyD88), TIR domain-containing adaptor inducing interferon (TRIF) and p65 NF-κB subunit protein levels. Exercise also resulted in increased mRNA and protein expression of interleukin (IL)-6, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α. Proinflammatory effects of exercise were prevented by the administration of diclofenac, which blunted the activation of the TLR4/NF-κB pathway and the inflammatory response in the liver of exercised rats. Results from the present study highlight the role of TLR4 as a target for anti-inflammatory interventions. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

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Patricia Dillenburg-Pilla

Full Text Available Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

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Garcia, Neus; Tomàs, Marta; Santafé, Manel M; Besalduch, Nuria; Lanuza, Maria A; Tomàs, Josep

2010-12-08

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C₂₇H₂₁N₃O₅)] or p75(NTR) [Pep5 (C₈₆H₁₁₁N₂₅O₁₉S₂] signaling. The effect of BDNF depends on the M₁ and M₂ muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M₁- and M₂-subtype muscarinic pathways.

Lupus risk variants in the PXK locus alter B-cell receptor internalization

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Samuel E. Vaughn

2015-01-01

Full Text Available Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus, rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb common haplotype that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3’ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 x 10-10, OR 0.81 (0.75 – 0.86. Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 200kb.Functionally, we found that PXK operates on the B-cell antigen receptor (BCR; we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.

The exhausted CD4+CXCR5+ T cells involve the pathogenesis of human tuberculosis disease.

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Bosco, Munyemana Jean; Wei, Ming; Hou, Hongyan; Yu, Jing; Lin, Qun; Luo, Ying; Sun, Ziyong; Wang, Feng

2018-06-21

The CD4 + CXCR5 + T cells have been previously established. However, their decreased frequency during tuberculosis (TB) disease is partially understood. The aim of this study was to explore the depletion of CD4 + CXCR5 + T cells in human TB. The frequency and function of CD4 + CXCR5 + T cells were evaluated in active TB (ATB) patients and healthy control (HC) individuals. The function of CD4 + CXCR5 + T cells was determined after blockade of inhibitory receptors. The frequency of CD4 + CXCR5 + T cells was decreased in ATB patients. The expression of activation markers (HLA-DR and ICOS) and inhibitory receptors (Tim-3 and PD-1) on CD4 + CXCR5 + T cells was increased in ATB group. TB-specific antigen stimulation induced higher expression of inhibitory receptors than phytohemagglutinin stimulation in ATB group. In contrast, TB antigen stimulation did not induce a significantly increased expression of IL-21 and Ki-67 on CD4 + CXCR5 + T cells. However, blockade of inhibitory receptors Tim-3 and PD-1 not only increased the frequency of CD4 + CXCR5 + T cells, but also restored their proliferation and cytokine secretion potential. An increased expression of inhibitory receptors involves the depletion of CD4 + CXCR5 + T cells, and blockade of inhibitory receptors can restore the function of CD4 + CXCR5 + T cells in ATB patients. Copyright © 2018. Published by Elsevier Ltd.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

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Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Nuclear receptor 4A (NR4A) family - orphans no more.

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Safe, Stephen; Jin, Un-Ho; Morpurgo, Benjamin; Abudayyeh, Ala; Singh, Mandip; Tjalkens, Ronald B

2016-03-01

The orphan nuclear receptors NR4A1, NR4A2 and NR4A3 are immediate early genes induced by multiple stressors, and the NR4A receptors play an important role in maintaining cellular homeostasis and disease. There is increasing evidence for the role of these receptors in metabolic, cardiovascular and neurological functions and also in inflammation and inflammatory diseases and in immune functions and cancer. Despite the similarities of NR4A1, NR4A2 and NR4A3 and their interactions with common cis-genomic elements, they exhibit unique activities and cell-/tissue-specific functions. Although endogenous ligands for NR4A receptors have not been identified, there is increasing evidence that structurally-diverse synthetic molecules can directly interact with the ligand binding domain of NR4A1 and act as agonists or antagonists, and ligands for NR4A2 and NR4A3 have also been identified. Since NR4A receptors are key factors in multiple diseases, there are opportunities for the future development of NR4A ligands for clinical applications in treating multiple health problems including metabolic, neurologic and cardiovascular diseases, other inflammatory conditions, and cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Natural Cytotoxicity Receptors: Pattern Recognition and Involvement of Carbohydrates

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Angel Porgador

2005-01-01

Full Text Available Natural cytotoxicity receptors (NCRs, expressed by natural killer (NK cells, trigger NK lysis of tumor and virus-infected cells on interaction with cell-surface ligands of these target cells. We have determined that viral hemagglutinins expressed on the surface of virus-infected cells are involved in the recognition by the NCRs, NKp44 and NKp46. Recognition of tumor cells by the NCRs NKp30 and NKp46 involves heparan sulfate epitopes expressed on the tumor cell membrane. Our studies provide new evidence for the identity of the ligands for NCRs and indicate that a broader definition should be applied to pathological patterns recognized by innate immune receptors. Since nonmicrobial endogenous carbohydrate structures contribute significantly to this recognition, there is an imperative need to develop appropriate tools for the facile sequencing of carbohydrate moieties.

B cell antigen receptor signaling and internalization are mutually exclusive events.

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Ping Hou

2006-07-01

Full Text Available Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

[Inhibition of glycogen synthase kinase 3b activity regulates Toll-like receptor 4-mediated liver inflammation].

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Ren, Feng; Zhang, Hai-yan; Piao, Zheng-fu; Zheng, Su-jun; Chen, Yu; Chen, De-xi; Duan, Zhong-ping

2012-09-01

To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4). C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively. The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro

Low density lipoprotein induces upregulation of vasoconstrictive endothelin type B receptor expression

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Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

2014-01-01

Vasoconstrictive endothelin type B (ET(B)) receptors promote vasospasm and ischemic cerebro- and cardiovascular diseases. The present study was designed to examine if low density lipoprotein (LDL) induces upregulation of vasoconstrictive ET(B) receptor expression and if extracellular signal...

Histamine H4 receptor in oral lichen planus.

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Salem, A; Al-Samadi, A; Stegajev, V; Stark, H; Häyrinen-Immonen, R; Ainola, M; Hietanen, J; Konttinen, Y T

2015-04-01

Oral lichen planus (OLP) is an autoimmune disease characterized by a band-like T-cell infiltrate below the apoptotic epithelial cells and degenerated basement membrane. We tested the hypothesis that the high-affinity histamine H4 receptors (H4 Rs) are downregulated in OLP by high histamine concentrations and proinflammatory T-cell cytokines. Immunohistochemistry and immunofluorescence staining, image analysis and quantitative real-time polymerase chain reaction of tissue samples and cytokine-stimulated cultured SCC-25 and primary human oral keratinocytes. H4 R immunoreactivity was weak in OLP and characterized by mast cell (MC) hyperplasia and degranulation. In contrast to controls, H4 R immunostaining and MC counts were negatively correlated in OLP (P = 0.003). H4 R agonist at nanomolar levels led to a rapid internalization of H4 Rs, whereas high histamine concentration and interferon-γ decreased HRH4 -gene transcripts. Healthy oral epithelial cells are equipped with H4 R, which displays a uniform staining pattern in a MC-independent fashion. In contrast, in OLP, increased numbers of activated MCs associate with increasing loss of epithelial H4 R. Cell culture experiments suggest a rapid H4 R stimulation-dependent receptor internalization and a slow cytokine-driven decrease in H4 R synthesis. H4 R may be involved in the maintenance of healthy oral mucosa. In OLP, this maintenance might be impaired by MC degranulation and inflammatory cytokines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

GABA and its B-receptor are present at the node of Ranvier in a small population of sensory fibers, implicating a role in myelination

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Corell, Mikael; Wicher, Grzegorz; Radomska, Katarzyna J

2015-01-01

throughout development and after injury. A small population of myelinated sensory fibers displayed all of these molecules at the node of Ranvier, indicating a role in axon-glia communication. Functional studies using GABAB receptor agonists and antagonists were performed in fetal DRG primary cultures...... to study the function of this receptor during development. The results show that GABA, via its B receptor, is involved in the myelination process but not in Schwann cell proliferation. The data from adult nerves suggest additional roles in axon-glia communication after injury.......The γ-aminobutyric acid (GABA) type B receptor has been implicated in glial cell development in the peripheral nervous system (PNS), although the exact function of GABA signaling is not known. To investigate GABA and its B receptor in PNS development and degeneration, we studied the expression...

Identification of a novel stereotypic IGHV4-59/IGHJ5-encoded B-cell receptor subset expressed by various B-cell lymphomas with high affinity rheumatoid factor activity

NARCIS (Netherlands)

Bende, Richard J.; Janssen, Jerry; Wormhoudt, Thera A. M.; Wagner, Koen; Guikema, Jeroen E. J.; van Noesel, Carel J. M.

2016-01-01

Subsets of mucosa-associated lymphoid tissue (MALT) lymphoma, splenic marginal zone B-cell lymphoma (MZBCL) and chronic lymphocytic leukemia (CLL) have been identified that express near-identical B-cell receptors (BCRs), strongly suggesting selection by restricted antigenic epitopes. We here report

The effect of melatonin from slow-release implants on basic and TLR-4-mediated gene expression of inflammatory cytokines and their receptors in the choroid plexus in ewes.

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Kowalewska, M; Herman, A P; Szczepkowska, A; Skipor, J

2017-08-01

The present study concerns the effect of melatonin from slow-release implants on the expression of genes coding interleukin-1β (Il1B), inerleukin-6 (Il6), tumour necrosis factor α (Tnf) and their receptors: IL-1 receptor type I (Il1r1) and type II (Il1r2), IL-6 receptor (Il6r) and signal transducer (Il6st), TNFα receptor type I (Tnfrsf1a) and II (Tnfrsf1b) and retinoid-related orphan receptor α (RorA) and Rev.-erbα in the ovine choroid plexus (CP) under basal and lipopolysaccharide (LPS)-challenged conditions. Studies were performed on four groups: 1) sham-implanted and placebo-treated, 2) melatonin-implanted (Melovine, 18mg) and placebo-treated, 3) sham-implanted and LPS-treated (400ng/kg of body weight) and 4) melatonin-implanted and LPS-treated. Under basal conditions, we observed weak expression of Tnf, low expression of Il1B, Il6 and Il1r2 and intermediate expression of other cytokines receptors. LPS treatment induced (P≤0.05) expression in all cytokines and their receptors, except Il6r 3h after the administration. Melatonin attenuated (P≤0.05) LPS-induced up-regulation of Il6 but had no effect on other cytokines and their receptors and up-regulated (P≤0.05) Rev.-erbα expression under basal conditions. This indicates that melatonin from slow-release implants suppresses TLR4-mediated Il6 expression in the ovine CP via a mechanism likely involving clock genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain.

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Chen, Lei; Liu, Jin-cheng; Zhang, Xiao-nan; Guo, Yan-yan; Xu, Zhao-hui; Cao, Wei; Sun, Xiao-li; Sun, Wen-ji; Zhao, Ming-Gao

2008-06-01

Gentiopicroside is one of the secoiridoid compound isolated from Gentiana lutea. It exhibits analgesic activities in the mice. The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission and induce glutamate NMDA NR2B receptor expression in the ACC. But little is known about Gentiopicroside on the persistent inflammatory pain and chronic pain-induced synaptic transmission changes in the ACC. The present study was undertaken to investigate its analgesic activities and central synaptic modulation to the peripheral painful inflammation. Gentiopicroside produced significant analgesic effects against persistent inflammatory pain stimuli in mice. Systemic administration of Gentiopicroside significantly reversed NR2B over-expression during the chronic phases of persistent inflammation caused by hind-paw administration of complete Freunds adjuvant (CFA) in mice. Whole-cell patch clamp recordings revealed that Gentiopicroside significantly reduced NR2B receptors mediated postsynaptic currents in the ACC. Our findings provide strong evidence that analgesic effects of Gentiopicroside involve down-regulation of NR2B receptors in the ACC to persistent inflammatory pain.

The α4β2 nicotine acetylcholine receptor agonist ispronicline induces c-Fos expression in selective regions of the rat forebrain

DEFF Research Database (Denmark)

Jacobsen, Julie; Hansen, Henrik H; Kiss, Alexander

2012-01-01

The dominant nicotine acetylcholine receptor (nAChR) subtype in the brain is the pentameric receptor containing both α4 and β2 subunits (α4β2). Due to the lack of selective agonists it has not been ruled out what neuronal circuits that are stimulated after systemic administration with nicotine. We...... or indirectly involved in acute stress regulation after a single dose of ispronicline, supports earlier studies that the α4β2 receptors are strongly involved in nicotine-dependent activation of the hypothalamo-pituitary adrenocortical axis....

Serotonin 1B Receptors Regulate Prefrontal Function by Gating Callosal and Hippocampal Inputs

DEFF Research Database (Denmark)

Kjaerby, Celia; Athilingam, Jegath; Robinson, Sarah E

2016-01-01

Both medial prefrontal cortex (mPFC) and serotonin play key roles in anxiety; however, specific mechanisms through which serotonin might act on the mPFC to modulate anxiety-related behavior remain unknown. Here, we use a combination of optogenetics and synaptic physiology to show that serotonin...... acts presynaptically via 5-HT1B receptors to selectively suppress inputs from the contralateral mPFC and ventral hippocampus (vHPC), while sparing those from mediodorsal thalamus. To elucidate how these actions could potentially regulate prefrontal circuit function, we infused a 5-HT1B agonist...... into the mPFC of freely behaving mice. Consistent with previous studies that have optogenetically inhibited vHPC-mPFC projections, activating prefrontal 5-HT1B receptors suppressed theta-frequency mPFC activity (4-12 Hz), and reduced avoidance of anxiogenic regions in the elevated plus maze. These findings...

Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

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Sebastian König

Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster.

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Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

2017-01-01

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster , a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT 1A , Dm5-HT 1B , and Dm5-HT 7 couple to cAMP signaling cascades, the Dm5-HT 2A receptor leads to Ca 2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT 2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT 2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT 2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT 2B 's pharmacology, we evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT 2B signaling in vitro and in vivo .

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

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Sato, Masaki; Ogura, Kazuhiro; Kimura, Maki; Nishi, Koichi; Ando, Masayuki; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2018-04-27

Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 -negative medium-sized neurons. Action potentials in these isolectin B 4 -negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential

A Novel Role of Serotonin Receptor 2B Agonist as an Anti-Melanogenesis Agent

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Eun Ju Oh

2016-04-01

Full Text Available BW723C86, a serotonin receptor 2B agonist, has been investigated as a potential therapeutic for various conditions such as anxiety, hyperphagia and hypertension. However, the functional role of BW723C86 against melanogenesis remains unclear. In this study, we investigate the effect of serotonin receptor 2B (5-HTR2B agonist on melanogenesis and elucidate the mechanism involved. BW723C86 reduced melanin synthesis and intracellular tyrosinase activity in melan-A cells and normal human melanocytes. The expression of melanogenesis-related proteins (tyrosinase, TRP-1 and TRP-2 and microphthalmia-associated transcription factor (MITF in melan-A cells decreased after BW723C86 treatment. The promoter activity of MITF was also reduced by BW723C86 treatment. The reduced level of MITF was associated with inhibition of protein kinase A (PKA and cAMP response element-binding protein (CREB activation by BW723C86 treatment. These results suggest that the serotonin agonist BW723C86 could be a potential therapeutic agent for skin hyperpigmentation disorders.

Kinin B1 Receptor Promotes Neurogenic Hypertension Through Activation of Centrally Mediated Mechanisms.

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Sriramula, Srinivas; Lazartigues, Eric

2017-12-01

Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension. © 2017 American Heart Association, Inc.

An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.

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Carmen H Coxon

Full Text Available Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM and an immunoreceptor tyrosine-based switch motif (ITSM. The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50 for both CRP and collagen.

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

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Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transient receptor potential vanilloid 1 (TRPV1), TRPV4, and the kidney

DEFF Research Database (Denmark)

Kassmann, M.; Harteneck, C.; Zhu, Z.

2013-01-01

Recent preclinical data indicate that activators of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) may improve the outcome of ischaemic acute kidney injury (AKI). The underlying mechanisms are unclear, but may involve TRPV1 channels in dorsal root ganglion neuro...... pharmacological TRPV modulators may be a successful strategy for better treatment of acute or chronic kidney failure.......-activated potassium channels and promote vasodilation. The TRPV receptors can also form heteromers that exhibit unique conductance and gating properties, further increasing their spatio-functional diversity. This review summarizes data on electrophysiological properties of TRPV1/4 and their modulation by endogenous...

The role of GluN2B-containing NMDA receptors in short- and long-term fear recall.

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Mikics, Eva; Toth, Mate; Biro, Laszlo; Bruzsik, Biborka; Nagy, Boglarka; Haller, Jozsef

2017-08-01

N-methyl-d-aspartate (NMDA) receptors are crucial synaptic elements in long-term memory formation, including the associative learning of fearful events. Although NMDA blockers were consistently shown to inhibit fear memory acquisition and recall, the clinical use of general NMDA blockers is hampered by their side effects. Recent studies revealed significant heterogeneity in the distribution and neurophysiological characteristics of NMDA receptors with different GluN2 (NR2) subunit composition, which may have differential role in fear learning and recall. To investigate the specific role of NMDA receptor subpopulations with different GluN2 subunit compositions in the formation of lasting traumatic memories, we contrasted the effects of general NMDA receptor blockade with GluN2A-, GluN2B-, and GluN2C/D subunit selective antagonists (MK-801, PEAQX, Ro25-6981, PPDA, respectively). To investigate acute and lasting consequences, behavioral responses were investigated 1 and 28days after fear conditioning. We found that MK-801 (0.05 and 0.1mg/kg) decreased fear recall at both time points. GluN2B receptor subunit blockade produced highly similar effects, albeit efficacy was somewhat smaller 28days after fear conditioning. Unlike MK-801, Ro25-6981 (3 and 10mg/kg) did not affect locomotor activity in the open-field. In contrast, GluN2A and GluN2C/D blockers (6 and 20mg/kg PEAQX; 3 and 10mg/kg PPDA, respectively) had no effect on conditioned fear recall at any time point and dose. This sharp contrast between GluN2B- and other subunit-containing NMDA receptor function indicates that GluN2B receptor subunits are intimately involved in fear memory formation, and may provide a novel pharmacological target in post-traumatic stress disorder or other fear-related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Large vessel involvement by IgG4-related disease

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Perugino, Cory A.; Wallace, Zachary S.; Meyersohn, Nandini; Oliveira, George; Stone, James R.; Stone, John H.

2016-01-01

Abstract Objectives: IgG4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs and lead to tumefactive, tissue-destructive lesions. Reports have described inflammatory aortitis and periaortitis, the latter in the setting of retroperitoneal fibrosis (RPF), but have not distinguished adequately between these 2 manifestations. The frequency, radiologic features, and response of vascular complications to B cell depletion remain poorly defined. We describe the clinical features, radiology findings, and treatment response in a cohort of 36 patients with IgG4-RD affecting large blood vessels. Methods: Clinical records of all patients diagnosed with IgG4-RD in our center were reviewed. All radiologic studies were reviewed. We distinguished between primary large blood vessel inflammation and secondary vascular involvement. Primary involvement was defined as inflammation in the blood vessel wall as a principal focus of disease. Secondary vascular involvement was defined as disease caused by the effects of adjacent inflammation on the blood vessel wall. Results: Of the 160 IgG4-RD patients in this cohort, 36 (22.5%) had large-vessel involvement. The mean age at disease onset of the patients with large-vessel IgG4-RD was 54.6 years. Twenty-eight patients (78%) were male and 8 (22%) were female. Thirteen patients (36%) had primary IgG4-related vasculitis and aortitis with aneurysm formation comprised the most common manifestation. This affected 5.6% of the entire IgG4-RD cohort and was observed in the thoracic aorta in 8 patients, the abdominal aorta in 4, and both the thoracic and abdominal aorta in 3. Three of these aneurysms were complicated by aortic dissection or contained perforation. Periaortitis secondary to RPF accounted for 27 of 29 patients (93%) of secondary vascular involvement by IgG4-RD. Only 5 patients demonstrated evidence of both primary and secondary blood vessel involvement. Of those treated with

The MC4 receptor and control of appetite

NARCIS (Netherlands)

Adan, R. A. H.; Tiesjema, B.; Hillebrand, J. J. G.; La Fleur, S. E.; Kas, M. J. H.; de Krom, M.

2006-01-01

Mutations in the human melanocortin (MC)4 receptor have been associated with obesity, which underscores the relevance of this receptor as a drug target to treat obesity. Infusion of MC4R agonists decreases food intake, whereas inhibition of MC receptor activity by infusion of an MC receptor

β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

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Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

2014-01-01

Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

Variation in melatonin receptors (Mel(1a) and Mel(1b)) and androgen receptor (AR) expression in the spleen of a seasonally breeding bird, Perdicula asiatica.

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Yadav, S K; Haldar, C; Singh, S S

2011-12-01

Daily variation in the peripheral level of melatonin plays a major role in integrating reproduction and environmental information for seasonally breeding birds. However, the variation in immunity and reproduction has never been assessed in any avian species on a 24 h time scale. Therefore, to understand the relationship between immune function and reproductive phases in a seasonally breeding bird, Perdicula asiatica, the Indian jungle bush quail, we studied the daily variation of melatonin and testosterone levels along with expression of their receptors Mel(1a), Mel(1b), and androgen receptor in the spleen during the reproductively active phase. Immunocytochemistry for the melatonin receptors Mel(1a) and Mel(1b) presented a differential distribution pattern. Western blot of splenic protein suggested a daily rhythm of melatonin receptors, while acrophases for the two melatonin receptors Mel(1a) and Mel(1b) differed by 4 h, suggesting that the expression of the receptors may peak at different times, causing more of either Mel(1a) or Mel(1b) to be available at a particular time to mediate function. The circulatory melatonin level correlated with percentage stimulation ratio of splenocytes and plasma interleukin-2 level, but did not correlate with testosterone or androgen receptor, suggesting that melatonin could be a major hormone imparting a time-of-day effect on the modulation of immune function in a seasonally breeding bird during the reproductively active phase. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Hormone receptor densities in relation to 10B neutron capture therapy

International Nuclear Information System (INIS)

Hechter, O.; Schwartz, I.L.

1982-01-01

This presentation is a theoretical discussion of the possibility that appropriate steroid-carborane derivatives might be used to selectively deliver boron-10 ( 10 B) to tumor cells with sex-hormone receptors in sufficient concentration for effective neutron capture theory (NCT) of hormone-dependent mammary and prostatic cancer. The results indicate the concentrations of androgen receptors (AR) and progesterone receptors (PR) in malignant prostatic cells or of estrogen receptors (ER) in malignant mammary cells are two low to achieve nuclear 10 B concentrations of 1 + g per g of tumor by using a steroid ligand coupled to a single carborane cage

Upregulation of contractile endothelin type B receptors by lipid-soluble cigarette smoking particles in rat cerebral arteries via activation of MAPK

International Nuclear Information System (INIS)

Sandhu, Hardip; Xu, Cang Bao; Edvinsson, Lars

2010-01-01

Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ET B ) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) mediate the upregulation of contractile endothelin receptors in the cerebral arteries. Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSP with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-κB specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ET B receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ET B receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively. Results show that organ culture per se induced transcriptional upregulation of contractile ET B receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ET B receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ET B receptors. Thus, the MAPK-mediated upregulation of contractile ET B receptors in cerebral arteries might be a

Mannose receptor may be involved in small ruminant lentivirus pathogenesis

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Crespo Helena

2012-05-01

Full Text Available Abstract Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV of known genotype (A or B, and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR expression (transcript levels and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints. Control sheep were SRLV-seropositive asymptomatic (n = 3, seronegative (n = 3 or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case. MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively. The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.

Role of dopamine D4 receptors in copulatory behavior: Studies with selective D4 agonists and antagonists in male rats.

Science.gov (United States)

Sanna, Fabrizio; Contini, Andrea; Melis, Maria Rosaria; Argiolas, Antonio

2015-10-01

Dopamine influences the anticipatory and consummatory phases of sexual behavior, by acting on receptors of the D2 family (D2, D3 and D4) and in particular of the D2 subtype, although evidence for a role of D4 receptors in erectile function and copulatory behavior is also available. In order to clarify such a role of D4 receptors, the effect of selective D4 receptor agonists and antagonists on copulatory behavior of sexually potent male rats in classic copulation tests with a receptive female, was compared with that of apomorphine and haloperidol, a classic dopamine receptor agonist and antagonist, respectively. PD-168,077 (0.05-0.2mg/kg) and ABT-724 (0.01-0.04mg/kg), two selective D4 receptor agonists, given subcutaneously, improved dose-dependently copulatory behavior as shown by the decrease of mount frequency and post ejaculatory interval induced by PD-168,077, and of mount frequency, ejaculation latency, post ejaculatory and inter intromission intervals induced by ABT-724, and by the increase of ejaculation frequency and copulatory efficacy induced by both drugs. Conversely, L-745,870 (1-5mg/kg), a selective D4 receptor antagonist, given intraperitoneally, impaired dose-dependently copulatory behavior, as shown by the increase in intromission and ejaculation latencies, mount frequency, post ejaculatory interval and the decrease in ejaculation frequency and copulatory efficacy induced by this drug. L-745,870 (5mg/kg) administered before PD-168,077 (0.2mg/kg) or ABT-724 (0.04mg/kg), also abolished completely the facilitatory effects of both PD-168,077 and ABT-724 on sexual behavior. These results confirm the involvement of D4 receptors in specific aspects of male rat copulatory behavior that overlap only partially with those influenced by apomorphine and haloperidol. Copyright © 2015 Elsevier Inc. All rights reserved.

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

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Nguyen, Linda; Robson, Matthew J; Healy, Jason R; Scandinaro, Anna L; Matsumoto, Rae R

2014-01-01

Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [(3)H](+)-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients.

Science.gov (United States)

Marshall-Gradisnik, Sonya; Johnston, Samantha; Chacko, Anu; Nguyen, Thao; Smith, Peter; Staines, Donald

2016-12-01

Objective The pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is unknown; however, a small subgroup of patients has shown muscarinic antibody positivity and reduced symptom presentation following anti-CD20 intervention. Given the important roles of calcium (Ca 2+ ) and acetylcholine (ACh) signalling in B cell activation and potential antibody development, we aimed to identify relevant single nucleotide polymorphisms (SNPs) and genotypes in isolated B cells from CFS/ME patients. Methods A total of 11 CFS/ME patients (aged 31.82 ± 5.50 years) and 11 non-fatigued controls (aged 33.91 ± 5.06 years) were included. Flow cytometric protocols were used to determine B cell purity, followed by SNP and genotype analysis for 21 mammalian TRP ion channel genes and nine mammalian ACh receptor genes. SNP association and genotyping analysis were performed using ANOVA and PLINK analysis software. Results Seventy-eight SNPs were identified in nicotinic and muscarinic acetylcholine receptor genes in the CFS/ME group, of which 35 were in mAChM3. The remaining SNPs were identified in nAChR delta (n = 12), nAChR alpha 9 (n = 5), TRPV2 (n = 7), TRPM3 (n = 4), TRPM4 (n = 1) mAChRM3 2 (n = 2), and mAChRM5 (n = 3) genes. Nine genotypes were identified from SNPs in TRPM3 (n = 1), TRPC6 (n = 1), mAChRM3 (n = 2), nAChR alpha 4 (n = 1), and nAChR beta 1 (n = 4) genes, and were located in introns and 3' untranslated regions. Odds ratios for these specific genotypes ranged between 7.11 and 26.67 for CFS/ME compared with the non-fatigued control group. Conclusion This preliminary investigation identified a number of SNPs and genotypes in genes encoding TRP ion channels and AChRs from B cells in patients with CFS/ME. These may be involved in B cell functional changes, and suggest a role for Ca 2+ dysregulation in AChR and TRP ion channel signalling in the pathomechanism of CFS/ME.

Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

DEFF Research Database (Denmark)

Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao

2006-01-01

In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA an...

The roles of Eph receptors in contextual fear conditioning memory formation.

Science.gov (United States)

Dines, Monica; Grinberg, Svetlana; Vassiliev, Maria; Ram, Alon; Tamir, Tal; Lamprecht, Raphael

2015-10-01

Eph receptors regulate glutamate receptors functions, neuronal morphology and synaptic plasticity, cellular events believed to be involved in memory formation. In this study we aim to explore the roles of Eph receptors in learning and memory. Toward that end, we examined the roles of EphB2 and EphA4 receptors, key regulators of synaptic functions, in fear conditioning memory formation. We show that mice lacking EphB2 (EphB2(-/-)) are impaired in short- and long-term contextual fear conditioning memory. Mice that express a carboxy-terminally truncated form of EphB2 that lacks forward signaling, instead of the full EphB2, are impaired in long-term, but not short-term, contextual fear conditioning memory. Long-term contextual fear conditioning memory is attenuated in CaMKII-cre;EphA4(lx/-) mice where EphA4 is removed from all pyramidal neurons of the forebrain. Mutant mice with targeted kinase-dead EphA4 (EphA4(KD)) exhibit intact long-term contextual fear conditioning memory showing that EphA4 kinase-mediated forward signaling is not needed for contextual fear memory formation. The ability to form long-term conditioned taste aversion (CTA) memory is not impaired in the EphB2(-/-) and CaMKII-cre;EphA4(lx/-) mice. We conclude that EphB2 forward signaling is required for long-term contextual fear conditioning memory formation. In contrast, EphB2 mediates short-term contextual fear conditioning memory formation in a forward signaling-independent manner. EphA4 mediates long-term contextual fear conditioning memory formation in a kinase-independent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

Science.gov (United States)

Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

2012-03-01

Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice.

Science.gov (United States)

Wu, Gang; Liu, Xiu-Xiu; Lu, Nan-Nan; Liu, Qi-Bing; Tian, Yun; Ye, Wei-Feng; Jiang, Guo-Jun; Tao, Rong-Rong; Han, Feng; Lu, Ying-Mei

2017-06-01

The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain. Here, we show that the endothelial cell-specific deletion of ErbB4 induces decreased exploratory behavior in adult mice. However, the water maze task for spatial memory and the memory reconsolidation test reveal no changes; additionally, we observe no impairment in CaMKII phosphorylation in Cdh5Cre;ErbB4 f/f mice, which indicates that the endothelial ErbB4 deficit leads to decreased exploratory activity rather than direct memory deficits. Furthermore, decreased brain metabolism, which was measured using micro-positron emission tomography, is observed in the Cdh5Cre;ErbB4 f/f mice. Consistently, the immunoblot data demonstrate the downregulation of brain Glut1, phospho-ULK1 (Ser555), and TIGAR in the endothelial ErbB4 conditional knockout mice. Collectively, our findings suggest that endothelial ErbB4 plays a critical role in regulating brain function, at least in part, through maintaining normal brain energy homeostasis. Targeting ErbB4 or the modulation of endothelial ErbB4 signaling may represent a rational pharmacological approach to treat neurological disorders. © 2017 John Wiley & Sons Ltd.

Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

Energy Technology Data Exchange (ETDEWEB)

Ngaotepprutaram, Thitirat [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Kaplan, Barbara L.F. [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Neuroscience Program, Michigan State University (United States); Kaminski, Norbert E., E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States)

2013-11-15

We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

Science.gov (United States)

Peñas-Cazorla, Raúl; Vilaró, M Teresa

2015-11-01

Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

Enhancement of bradykinin and resensitization of its B2 receptor.

Science.gov (United States)

Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

1999-03-01

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

International Nuclear Information System (INIS)

Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E.

1991-01-01

The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response

International Nuclear Information System (INIS)

Icli, Basak; Bharti, Ajit; Pentassuglia, Laura; Peng, Xuyang; Sawyer, Douglas B.

2012-01-01

Highlights: ► ErbB4 localizes to cardiac myocyte nuclei as a full-length receptor. ► Cardiac myocytes express predominantly JM-a/CYT-1 ErbB4. ► Myocyte p53 activation in response to doxorubicin requires ErbB4 activity. -- Abstract: The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

Directory of Open Access Journals (Sweden)

Nicholas K H Ostan

2017-03-01

Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

Expression of ET(A) and ET(B) receptor mRNA in human cerebral arteries

DEFF Research Database (Denmark)

Hansen-Schwartz, J; Szok, D; Edvinsson, L

2002-01-01

The vascular effects of endothelins (ET) are in mammals mediated via two receptor subtypes, endothelin A (ET(A), mainly constrictive) and endothelin B (ET(B), mainly dilating) receptors. We have examined the presence of ET(A) and ET(B) receptor mRNA using the reverse transcription polymerase chai...

Tumor Suppressor Activity of the EphB2 Receptor in Prostate Cancer

National Research Council Canada - National Science Library

Pasquale, Elena B

2007-01-01

Mutations have been recently identified in the EphB2 receptor gene in prostate cancer suggesting that EphB2, a member of the large Eph receptor tyrosine kinase family, is a tumor suppressor in prostate cancer...

Tumor Suppressor Activity of the EphB2 Receptor in Prostate Cancer

National Research Council Canada - National Science Library

Pasquale, Elena B

2006-01-01

Mutations have been recently identified in the EphB2 receptor gene in prostate cancer suggesting that EphB2, a member of the large Eph receptor tyrosine kinase family, is a tumor suppressor in prostate cancer...

Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

Science.gov (United States)

Lee, Jin Ju; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Lee, Hu Jang; Min, Wongi; Her, Moon; Rhee, Man Hee; Watarai, Masahisa; Chang, Hong Hee; Kim, Suk

2016-04-21

Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

Involvement of AMPA receptors in the antidepressant-like effects of dextromethorphan in mice.

Science.gov (United States)

Nguyen, Linda; Matsumoto, Rae R

2015-12-15

Dextromethorphan (DM) is an antitussive with rapid acting antidepressant potential based on pharmacodynamic similarities to ketamine. Building upon our previous finding that DM produces antidepressant-like effects in the mouse forced swim test (FST), the present study aimed to establish the antidepressant-like actions of DM in the tail suspension test (TST), another well-established model predictive of antidepressant efficacy. Additionally, using the TST and FST, we investigated the role of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in the antidepressant-like properties of DM because accumulating evidence suggests that AMPA receptors play an important role in the pathophysiology of depression and may contribute to the efficacy of antidepressant medications, including that of ketamine. We found that DM displays antidepressant-like effects in the TST similar to the conventional and fast acting antidepressants characterized by imipramine and ketamine, respectively. Moreover, decreasing the first-pass metabolism of DM by concomitant administration of quinidine (CYP2D6 inhibitor) potentiated antidepressant-like actions, implying DM itself has antidepressant efficacy. Finally, in both the TST and FST, pretreatment with the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide) significantly attenuated the antidepressant-like behavior elicited by DM. Together, the data show that DM exerts antidepressant-like actions through AMPA receptors, further suggesting DM may act as a safe and effective fast acting antidepressant drug. Copyright © 2015 Elsevier B.V. All rights reserved.

The Role of Leukotriene B4 in Allergic Diseases

Directory of Open Access Journals (Sweden)

Hiroshi Ohnishi

2008-01-01

Full Text Available Leukotriene B4 (LTB4 is a lipid mediator with potent chemoattractant properties and that is rapidly generated from activated innate immune cells such as neutrophils, macrophages, and mast cells. Elevated levels of LTB4 have been reported in various allergic diseases and these levels have been related to disease activity and response to treatment. Recent studies using LTB4 receptor-1 (BLT1 antagonists or BLT1-deficient mice have revealed that ligation of BLT1 by LTB4 is important for the activation and recruitment of inflammatory cells including neutrophils, eosinophils, monocytes/macrophages, mast cells, dendritic cells, and more recently, effector T cells to inflamed tissues in various inflammatory diseases. The LTB4/BLT1 pathway appears to play an important role in the pathogenesis of severe persistent asthma, aspirin- and exercise-induced asthma, allergic rhinitis, and atopic dermatitis together with other mediators including cysteinyl leukotrienes, cytokines, and chemokines. LTB4 production is in general resistant to corticosteroid treatment. In fact, corticosteroids can upregulate BLT1 expression on corticosteroid-resistant inflammatory cells such as neutrophils, monocytes, and effector memory CD8+ T cells. As a result, this corticosteroid-resistant LTB4/BLT1 pathway may contribute to the development of inflammation in allergic diseases that do not respond to the introduction of corticosteroids. Inhibition of this pathway has potential therapeutic benefit in various allergic diseases that have involvement of corticosteroid-insensitivity.

Reduction of GABA/sub B/ receptor binding induced by climbing fiber degeneration in the rat cerebellum

International Nuclear Information System (INIS)

Kato, K.; Fukuda, H.

1985-01-01

When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABA/sub B/ receptor binding determined with 3 H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the B/sub max/ of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABA/sub B/ sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABA/sub A/ and benzodiazepine receptor binding labelled with 3 H-muscimol and 3 H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABA/sub B/ sites but neither GABA/sub A/ nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals. 28 references, 4 figures, 2 tables

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

Science.gov (United States)

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

Directory of Open Access Journals (Sweden)

Linda Nguyen

Full Text Available Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1 receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047 were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [(3H](+-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

Science.gov (United States)

Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma

2018-04-30

Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

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Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Ophthalmic antihistamines and H1-H4 receptors.

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Wade, Laurie; Bielory, Leonard; Rudner, Shara

2012-10-01

Antihistamines exert pharmacologic effects by binding to four histamine receptors (H1-H4) at different affinities, producing variable effects depending on the receptor they predominantly bind to. This review's purpose is to determine the relative potency of antihistamines by comparing their binding affinities to these receptors. Studies on binding affinities of antihistamines to histamine receptors were reviewed and the dissociation constant for inhibitor binding (Ki) analyzed to determine the most and least potent antihistamine for each receptor. We retrieved the binding affinities for nineteen antihistamines. For H1 receptors, pyrilamine exhibited the highest affinity (Ki = 0.8 nM), and thioperamide the lowest (Ki = 280, 000 nM). For H2 receptors, ranitidine exhibited the highest affinity (Ki = 187 nM), and olopatadine the lowest (Ki = 100 ,000 nM). For the recently discovered H3 and H4 receptors, thioperamide exhibited the highest affinity (Ki = 1.1 nM), and olopatadine exhibited the lowest (Ki = 79 ,400 nM), to H3. Data on binding affinities to the H4 receptor exist for: ketotifen, pheniramine, ranitidine, cimetidine and thioperamide. Of these, thioperamide exhibited the highest affinity (Ki = 27 nM), whereas cimetidine and ranitidine exhibited the lowest affinity (Ki = >10, 000 nM) for H4 receptors. This review summarizes the relative potency of antihistamines based on their binding affinities to the four histamine receptors. Although data on binding affinities of antihistamines to the H4 receptor are sparse, it is apparent that further research on these histamine subtypes may open new venues for more direct treatment with a higher therapeutic efficacy on allergic disorders including those affecting the ocular surface.

Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor

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Keegan Achsah D

2011-05-01

Full Text Available Abstract Background Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined. Results Allergen treatment and lung-specific vascular endothelial growth factor (VEGF expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly

Cell Surface Trafficking of TLR1 Is Differentially Regulated by the Chaperones PRAT4A and PRAT4B*

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Hart, Bryan E.; Tapping, Richard I.

2012-01-01

The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression. PMID:22447933

Hydrostatic pressure and muscarinic receptors are involved in the release of inflammatory cytokines in human bladder smooth muscle cells.

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Liang, Zhou; Xin, Wei; Qiang, Liu; Xiang, Cai; Bang-Hua, Liao; Jin, Yang; De-Yi, Luo; Hong, Li; Kun-Jie, Wang

2017-06-01

Abnormal intravesical pressure results in a series of pathological changes. We investigated the effects of hydrostatic pressure and muscarinic receptors on the release of inflammatory cytokines in rat and human bladder smooth muscle cells (HBSMCs). Animal model of bladder outlet obstruction was induced by urethra ligation. HBSMCs were subjected to elevated hydrostatic pressure and/or acetylcholine (Ach). Macrophage infiltration in the bladder wall was determined by immunohistochemical staining. The expression of inflammatory genes was measured by RT-PCR, ELISA and immunofluorescence. In obstructed bladder, inflammatory genes and macrophage infiltration were remarkably induced. When HBSMCs were subjected to 200-300 cm H 2 O pressure for 2-24 h in vitro, the expressions of IL-6 and RANTES were significantly increased. Hydrostatic pressure promoted the protein levels of phospho-NFκB p65 and phospho-ERK1/2 as well as muscarinic receptors. Moreover, NFκB or ERK1/2 inhibitors suppressed pressure-induced inflammatory genes mRNA. When cells were treated with 1 μM acetylcholine for 6 h, a significant increase in IL-6 mRNA expression was detected. Acetylcholine also enhanced pressure-induced phospho-NFκB p65 and IL-6 protein expression. Additionally, pressure-induced IL-6 was partially suppressed by muscarinic receptors antagonists. Hydrostatic pressure and muscarinic receptors were involved in the secretion of inflammatory cytokines in HBSMCs, indicating a pro-inflammatory effect of the two factors in the pathological process of BOO. © 2016 Wiley Periodicals, Inc.

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

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Pfistershammer, Katharina; Klauser, Christoph; Pickl, Winfried F; Stöckl, Johannes; Leitner, Judith; Zlabinger, Gerhard; Majdic, Otto; Steinberger, Peter

2006-05-01

The B7 family member programmed-death-1-ligand 2 (PD-L2/B7-DC) is a ligand for programmed-death-receptor 1 (PD-1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD-L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD-L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane-bound anti-CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD-L2 to consistently inhibit T cell activation. PD-L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD-1 reverted the inhibitory effect of PD-L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD-L2 in murine systems. In line with our functional data that do not point to stimulatory PD-L2-ligands, we show that binding of PD-L2-immunoglobulin to activated human T cells is abrogated by PD-1 antibodies. Our results demonstrate that PD-L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.

AMP and adenosine are both ligands for adenosine 2B receptor signaling.

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Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

2018-01-15

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cysteinyl leukotrienes C4 and D4 downregulate human mast cell expression of toll-like receptors 1 through 7.

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Karpov, V; Ilarraza, R; Catalli, A; Kulka, M

2018-01-01

Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid molecules that mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability. As a result, CysLT receptor antagonists (LRA), such as montelukast, have been used to effectively treat patients with asthma. We have recently shown that mast cells are necessary modulators of innate immune responses to bacterial infection and an important component of this innate immune response may involve the production of CysLT. However, the effect of LRA on innate immune receptors, particularly on allergic effector cells, is unknown. This study determined the effect of CysLT on toll-like receptor (TLR) expression by the human mast cell line LAD2. Real-time PCR analysis determined that LTC4, LTD4 and LTE4 downregulated mRNA expression of several TLR. Specifically in human CD34+-derived human mast cells (HuMC), LTC4 inhibited expression of TLR1, 2, 4, 5, 6 and 7 while LTD4 inhibited expression of TLR1-7. Montelukast blocked LTC4-mediated downregulation of all TLR, suggesting that these effects were mediated by activation of the CysLT1 receptor (CysLT1R). Flow cytometry analysis confirmed that LTC4 downregulated surface expression of TLR2 which was blocked by montelukast. These data show that CysLT can modulate human mast cell expression of TLR and that montelukast may be beneficial for innate immune responses mediated by mast cells.

The prognostic value of Her4 receptor isoform expression in triple-negative and Her2 positive breast cancer patients

International Nuclear Information System (INIS)

Machleidt, Anna; Buchholz, Stefan; Diermeier-Daucher, Simone; Zeman, Florian; Ortmann, Olaf; Brockhoff, Gero

2013-01-01

Not only four but rather seven different human epidermal growth factor receptor related (Her) receptor tyrosine kinases (RTKs) have been described to be expressed in a variety of normal and neoplastic tissues: Her1, Her2, Her3, and additionally four Her4 isoforms have been identified. A differential expression of Her4 isoforms does not, however, play any role in either the molecular diagnostics or treatment decision for breast cancer patients. The prognostic and predictive impact of Her4 expression in breast cancer is basically unclear. We quantified the Her4 variants JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, and JM-b/CYT2 by isoform-specific polymerase chain reaction (qPCR) in (i) triple-negative, (ii) Her2 positive breast cancer tissues and (iii) in benign breast tissues. In all three tissue collectives we never found the JM-b/CYT1 or the JM-b/CYT2 isoform expressed. In contrast, the two JM-a/CYT1 and JM-a/CYT2 isoforms were always simultaneously expressed but at different ratios. We identified a positive prognostic impact on overall survival (OS) in triple-negative and event-free survival (EFS) in Her2 positive patients. This finding is independent of the absolute JM-a/CYT1 to JM-a/CYT2 expression ratio. In Her2 positive patients, Her4 expression only has a favorable effect in estrogen-receptor (ER)-positive but not in ER-negative individuals. In summary, JM-a/CYT1 and JM-a/CYT2 but not JM-b isoforms of the Her4 receptor are simultaneously expressed in both triple-negative and Her2 positive breast cancer tissues. Although different expression ratios of the two JM-a isoforms did not reveal any additional information, Her4 expression basically indicates a prolonged EFS and OFS. An extended expression analysis that takes all Her receptor homologs, including the Her4 isoforms, into account might render more precisely the molecular diagnostics required for the development of optimized targeted therapies

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

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Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

2015-01-01

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

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Wang, H Y; Paul, W E; Keegan, A D

1996-02-01

IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.