Membrane Expression Of Receptors Research Articles

Differential Membrane Expression of Toll-Like Receptors and Intracellular Cytokine Induction in Peripheral Blood Monocytes of Patients with Chronic Kidney Disease and Diabetic Nephropathy

Background: Toll-like receptors (TLRs) are key players in the innate immune system whose activation leads to an inflammatory response. Inflammation plays an important role in the pathogenesis of chronic kidney disease (CKD) and diabetes mellitus. The aim of our study was to assess the proinflammatory state of nondialysis CKD patients by evaluating the membrane expression of TLR2 and TLR4 and the intracellular IL-1β and IL-6 production in response to the ligand Pam3Cys-Ser-(Lys)4 (Pam3CSK4). Methods: 85 nondialysis CKD patients [mean estimated glomerular filtration rate: 34 (17-90) ml/min/1.73 m²] were divided in 2 groups: 55 nondiabetic CKD patients (CKD group) and 30 patients with diabetic nephropathy (DN group). The two groups were compared with 36 healthy subjects (control group). TLR2 and TLR4 membrane expression in monocytes and Pam3CSK4-induced intracellular production of IL-1β and IL-6 were assessed by flow cytometry. Results: Both patient groups showed increased TLR2 membrane expression compared with the control group, both at baseline (p < 0.05 for both) and after Pam3CSK4 stimulation (p < 0.05 for both). The DN group exhibited significantly higher TLR4 expression at baseline compared to the CKD and control groups (p < 0.04 and p < 0.02, respectively). Intracellular IL-1β and IL-6 levels at baseline were significantly lower in CKD patients compared to the DN and control groups. After Pam3CSK4 stimulation, intracellular IL-1β and IL-6 increased in all groups, but were lower in the CKD group versus the control group or DN group, which exhibited higher levels than the controls. Conclusions: Nondialysis CKD patients showed significant alterations in TLR2 and TLR4 membrane expression, and impaired Pam3CSK4-induced cytokine production in monocytes, a phenomenon that is markedly influenced by the presence of diabetes.

Abstract 13801: The p.Leu167del Mutation in APOE Gene causes Autosomal Dominant Hypercholesterolemia by Down-Regulation of LDL Receptor Expression in Hepatocytes

Introduction: The p.Leu167del mutation in APOE gene has been associated to different hyperlipidemias. However, the frequency of this mutation in autosomal dominant hypercholesterolemia and the mechanism of this association is not known. Objectives: To establish the frequency of p.Leu167del mutation in APOE gene in subjects with clinical diagnosis of Autosomal Dominant Hypercholesterolemia (ADH) non-dependent of LDLR, APOB nor PCSK9 genes, and to investigate the mechanism by which this mutation associates to ADH. Methods: We selected 288 unrelated subjects with ADH non-dependent of LDLR, APOB nor PCSK9 genes, and 220 unrelated normolipidemic subjects (control group). All available family members of mutation carrier subjects were also studied. Exon 4 of APOE gene was sequenced in all subjects. VLDL and LDL were isolated from p.Leu167del carriers and E3/E3 control subjects by ultracentrifugation. Quantification of lipoprotein uptake in HepG2 and THP-1 cells and LDL receptor membrane-expression in HepG2 cells were carried out by flow cytometry. Results: In the ADH group, 9 unrelated subjects (3.1%) were carriers of the p.Leu167del mutation in APOE gene and none in the control group. Eleven family members were carriers of the mutation, 7 with isolated hypercholesterolemia and 4 with mixed hyperlipidemia. A clear co-segregation of p.Leu167del mutation with elevated plasma cholesterol levels in the families was found. VLDL isolated from p.Leu167del mutation carriers had a significantly higher uptake by HepG2 and THP-1 cells compared to VLDL isolated from E3/E3 subjects (p&lt;0.01). When increasing incubation times of HepG2 cells with VLDL from p.Leu167del carriers, the LDL receptor expression in surface membrane diminished, in contrast with the VLDL from E3/E3 subjects. When pre-incubating HepG2 cells with VLDL from p.Leu167del mutation carriers, the percentage of LDL internalisation was significantly lower than when incubating with VLDL from E3/E3 subject (p&lt;0.01). Conclusion: The p.Leu167del mutation in APOE gene is associated to ADH in 3.1% of studied subjects. The mechanism of this association is higher uptake of VLDL, down regulation of the LDLR expression and decrease of LDL internalisation in hepatocytes.

Macrophage infection via selective capture of HIV-1-infected CD4+ T cells.

SummaryMacrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

Structural plasticity: mechanisms and contribution to developmental psychiatric disorders.

Synaptic plasticity mechanisms are usually discussed in terms of changes in synaptic strength. The capacity of excitatory synapses to rapidly modify the membrane expression of glutamate receptors in an activity-dependent manner plays a critical role in learning and memory processes by re-distributing activity within neuronal networks. Recent work has however also shown that functional plasticity properties are associated with a rewiring of synaptic connections and a selective stabilization of activated synapses. These structural aspects of plasticity have the potential to continuously modify the organization of synaptic networks and thereby introduce specificity in the wiring diagram of cortical circuits. Recent work has started to unravel some of the molecular mechanisms that underlie these properties of structural plasticity, highlighting an important role of signaling pathways that are also major candidates for contributing to developmental psychiatric disorders. We review here some of these recent advances and discuss the hypothesis that alterations of structural plasticity could represent a common mechanism contributing to the cognitive and functional defects observed in diseases such as intellectual disability, autism spectrum disorders and schizophrenia.

Contributions of purinergic P2X3 receptors within the midbrain periaqueductal gray to diabetes-induced neuropathic pain.

Hyperalgesia and allodynia are commonly observed in patients with diabetic neuropathy. The mechanisms responsible for neuropathic pain are not well understood. Thus, in this study, we examined the role played by purinergic P2X3 receptors of the midbrain periaqueductal gray (PAG) in modulating diabetes-induced neuropathic pain because this brain region is an important component of the descending inhibitory system to control central pain transmission. Our results showed that mechanical withdrawal thresholds were significantly increased by stimulation of P2X3 receptors in the dorsolateral PAG of rats (n = 12, P < 0.05 vs. vehicle control) using α,β-methylene-ATP (α,β-meATP, a P2X3 receptor agonist). In addition, diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) in rats, and mechanical allodynia was observed 3 weeks after STZ administration. Notably, the excitatory effects of P2X3 stimulation on mechanical withdrawal thresholds were significantly blunted in STZ-induced diabetic rats (n = 12, P < 0.05 vs. control animals) as compared with control rats (n = 12). Furthermore, the protein expression of P2X3 receptors in the plasma membrane of the dorsolateral PAG of STZ-treated rats was significantly decreased (n = 10, P < 0.05 vs. control animals) compared to that in control rats (n = 8), whereas the total expression of P2X3 receptors was not significantly altered. Overall, data of our current study suggest that a decrease in the membrane expression of P2X3 receptors in the PAG of diabetic rats is likely to impair the descending inhibitory system in modulating pain transmission and thereby contributes to the development of mechanical allodynia in diabetes.

Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory proteins

Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors.

Membrane expression of trail receptors DcR1 and DcR2 in the normal endometrium, endometrial atypical hyperplasia and endometrioid endometrial cancer

We aimed to evaluate the membrane expression of DcR1 and DcR2 in the normal endometrium (NE), endometrial atypical hyperplasia (EAH) and endometrioid endometrial cancer (EEC). The study comprised 101 patients: 20 NE, 14 EAH and 67 EEC. Membrane expression of DcR1 and DcR2 was examined and presented as total score (TS). The membrane expression of both DcR1 and DcR2 was more common in EEC than in NE (p < 0.001; p < 0.001). A strong correlation was found between type of endometrial tissue (NE/EAH/EEC) and the TS of DcR1 (p = 0.001) and DcR2 (p < 0.001). In EEC, the TS of DcR1 and DcR2 was not related to grading and survival. The TS of DcR1 negatively correlated with staging (p = 0.018), but DcR2 did not. The membrane expression of decoy receptors for TRAIL DcR1 and DcR2 is greater in NE than EEC. In EEC patients, membrane expression of DcR1 and DcR2 are not independent predictors of survival.

The C-terminal half of the α2C-adrenoceptor determines the receptor's membrane expression level and drug selectivity

In tissues as well as in transfected cells, α2C-adrenoceptors show poorer expression levels compared to α2A-adrenoceptors. In order to characterize which regions of the α2C-adrenoceptor are involved in regulating the expression of binding-competent receptors at the plasma membrane, six chimeric α2A-/α2C-adrenoceptors were constructed. The wild-type α2A- and α2C-adrenoceptors and the six chimeric α2A-/α2C-adrenoceptors were transiently transfected into human embryonic kidney 293 (HEK293) cells, and the expression levels were investigated by radioligand binding. The results show that the C-terminal half of the α2C-adrenoceptor, ranging from the second extracellular loop to the C-terminus, is the main determinant of the low expression level of binding-competent α2C-adrenoceptors in HEK293 cell membranes. The so-called retention signal in the N-terminus of the α2C-adrenoceptor had a less profound effect on the expression levels of the chimeric receptors. For seven drugs competing for [(3)H]-RX821002 binding, the K i values were determined at the wild-type α2A- and α2C-adrenoceptors and at four of the chimeric α2A-/α2C-adrenoceptors. The results show that the α2C- over α2A-selectivity of spiroxatrine, spiperone, clozapine, MK912, and chlorpromazine, as well as the α2A- over α2C-selectivity of BRL44408, resides mainly in the C-terminal half of the receptors. To some extent, the region comprising the N-terminal half of the receptors contributed to the α2C-selectivity of spiperone, clozapine, and chlorpromazine.

Abstract 465: Characterization of NK cell exhaustion in melanoma at different stages.

Abstract BACKGROUND: The concept of CD8+ T cell exhaustion in the context of metastatic cancer has been reinforced by the recent success of immunotherapies targeting the exhaustion markers CTLA-4 and PD-1 in advanced melanoma. Exhausted T cells are characterized by: 1) an over expression of inhibitory receptors or exhaustion markers such as CTLA-4, PD-1 and Tim-3; 2) a loss of function (cytotoxicity, cytokine production and proliferation); 3) a down-regulation of cytokine receptors, rendering them refractory to cytokine stimulation; and 4) changes in transcription factors, including downregulation of T-bet expression. T cell exhaustion has been extensively studied in the context of chronic infectious diseases and different types of cancer, however little is known about exhaustion of NK cells in the same background. NK cells, innate immune cells that eliminate tumors through cytotoxicity and IFN-γ production, have a key role in immune surveillance of tumors, including melanoma. In this study, we characterize and compare the phenotype of NK cells from patients with melanoma of different stages (I, II, III, IV) and healthy donors. METHODS: We compared NK cells from 10 patients with melanoma at each stage of presentation (I, II, III, IV) and from 20 healthy donors as it relates to membrane expression of activating (NKG2D and NKp46) and inhibitory receptors (KIRB1 and KIRNKAT2), function (cytotoxicity, IFN-γ production and proliferation) and the intracellular expression of the transcription factor T-bet. NK cells’ cytotoxicity was measured by Lamp-1 expression using K562 cells as target cells. IFN-γ production was measured after 4h stimulation with rhIL-12. Proliferation was quantified by CFSE after 6 days in the presence of rhIL-2. RESULTS: As the stage of the melanoma advanced we observed a pattern of gradual increase of inhibitory receptors (with a higher difference between stage II and stage III) and a concomitant decrease of activating receptors (with a higher difference between stage III and stage IV). With regard to the three most important NK cell functions, IFN-γ production and proliferation were impaired beginning with stage I and advancing to stage IV, while cytotoxicity was reduced only at stage III and stage IV. Similarly, T-bet expression also began to decrease at stage III. CONCLUSIONS: These data suggest that NK cells from melanoma patients gradually develop a phenotypic and functional profile consistent with exhaustion, beginning with stage I and advancing to stage IV. The exhaustion phenotype begins with initial loss of IFN-γ production and proliferation ability, and finally cytotoxicity. Therefore, the concept that NK cells also become exhausted make them an interesting population to target therapeutically. Furthermore, as the exhaustion phenotype is acquired progressively through the stages, and not exclusive to stage IV, it may be possible to intervene early in the course of tumor progression. Citation Format: Ines Pires Da Silva, Anne Gallois, Iman Osman, Nina Bhardwaj. Characterization of NK cell exhaustion in melanoma at different stages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2013-465

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Abstract 5180: RNAi-mediated diminution of myoferlin increases adhesion of breast cancer cells

Abstract Introduction: Ferlins are a family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including plasma membrane repair, regulation of membrane expression of receptors, and endocytosis. Dysferlin was recently reported to regulate platelet endothelial cellular adhesion molecule-1 (PECAM-1) in endothelial cells (1). We have shown that ablation of myoferlin (MYOF) in breast tumor cells attenuates invasion in vitro (in review, J Cell Sci). Moreover, loss of MYOF was found to be associated with reduced phosphorylation of a number of receptor tyrosine kinases (in press, PNAS), and others have reported on the role of MYOF in tyrosine kinase receptors expression at the plasma membrane (2). These results prompted us to hypothesize that MYOF may be involved in cell-matrix adhesion. Methods &amp; Results: We used lentiviral-mediated RNA interference (RNAi) gene silencing to generate stable MYOF-deficient MDA-MB-231 cells and confirmed MYOF reduction with immunoblotting and qRT-PCR. RNAi control cells were generated in tandem with a non-human gene targeting construct. Relative adhesion and spreading of the wild-type, control, and MYOF-deficient epithelial cells were evaluated using a real-time electrical impedance measurement system (xCELLigence, Roche Applied Science). Results show a significant increase in the adhesion and spreading of the MYOF-deficient cells in comparision to wild-type and control cells. A trypsin assay developed using the same electrical impedance system demonstrated greater adhesion strength in the MYOF-deficient cells. This was measured by the duration of time necessary to reduce the pre-trypsin-treatment impedance by one-half. These results were further validated using a conventional parallel plate device, in which MYOF-deficient cells were found to be more adherent than controls when subjected to a range of shear stresses (1-10 dynes/cm2). PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control MDA-MB-231 cells indicated that intercellular adhesion molecule 1 (ICAM-1/CD54) and vitronectin mRNA levels are increased following MYOF-diminution, and may potentially contribute to the mechanism behind the altered adhesion in MYOF-deficient cells. Conclusion: Myoferlin is important for the regulation of breast cancer epithelial cell adhesion. Since dysregulation of adhesion is often associated with cancer invasion and metastasis, myoferlin may play a role in these processes in breast carcinoma. (1) Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, et al. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 2010 Nov;30 (11):2196-204. (2) Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P. Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 2011 May 6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5180. doi:1538-7445.AM2012-5180

Interplay between ionotropic receptors modulates inhibitory synaptic strength

The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at a synapse determines neuronal outputs in the CNS. In a recent study, we describe that excitatory and inhibitory transmitter-gated channels physically crosstalk each other at the cellular and molecular level. Increased membrane expression of ATP P2X4 receptors by using an interference peptide competing with the intracellular endocytosis motif enhances neuronal excitability, which is further enhanced by reciprocal interaction between post-synaptic ATP- and GABA-gated channels. Molecular interaction is supported by experiments of co-immunoprecipitation and mutagenesis of P2X4 subunit. Two amino acids in the intracellular carboxyl tail of P2X4 subunit appears to be responsible for this crosstalk. Our recent study provides molecular and electrophysiological evidence for physical interaction between excitatory and inhibitory receptors that appears to be crucial in determining synaptic strength at central synapses.

Retrieval-specific endocytosis of GluA2-AMPARs underlies adaptive reconsolidation of contextual fear

Upon retrieval, fear memories are rendered labile and prone to modification, necessitating a restabilization process of reconsolidation to persist further. This process is also crucial for modulating both strength and content of an existing memory and forms a promising therapeutic target for fear-related disorders. However, the molecular and cellular mechanism of adaptive reconsolidation still remains obscure. Here we show that retrieval of fear memory induces a biphasic temporal change in GluA2-containing AMPA-type glutamate receptor (AMPAR) membrane expression and synaptic strength in the mouse dorsal hippocampus. Blockade of retrieval-induced, regulated, GluA2-dependent endocytosis enhanced subsequent expression of fear. In addition, this blockade prevented the loss of fear response after reconsolidation-update of fear memory content in the long-term. Thus, endocytosis of GluA2-containing AMPARs allows plastic changes at the synaptic level that exerts an inhibitory constraint on memory strengthening and underlies the loss of fear response by reinterpretation of memory content during adaptive reconsolidation.

The truncated ghrelin receptor polypeptide (GHS-R1b) is localized in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors (GHS-R1a) to attenuate their cell surface expression

The ghrelin receptor (GHS-R1a) is remarkable amongst G-protein-coupled receptors for its high degree of constitutive activity, and this agonist-independent activity may be important for its physiological function in the control of food intake and body weight. Ghrelin receptors form heterodimers with the truncated ghrelin receptor polypeptide (GHS-R1b), which has a dominant-negative effect on ghrelin receptor function. Here we show that GHS-R1b has an intracellular localization distinct from ghrelin receptors, being primarily localized in the endoplasmic reticulum. Immunocytochemical studies suggest that GHS-R1b decreases the plasma membrane expression of ghrelin receptors, but the overall distribution profile of ghrelin receptors in isolated subcellular fractions is unaffected by GHS-R1b. Using bioluminescence resonance energy transfer methods, we have shown that while ghrelin receptor homodimers are evenly distributed in all subcellular fractions, GHS-R1a/GHS-R1b heterodimers are concentrated within the endoplasmic reticulum and these results suggest that GHS-R1b traps ghrelin receptors within the endoplasmic reticulum by the process of oligomerization. Furthermore, ghrelin receptors constitutively activated extracellular signal-regulated kinases 1/2 in the endoplasmic reticulum, but this small response was not affected by GHS-R1b and its physiological relevance is uncertain. Taken together, these results suggest that ghrelin receptors can be retained in the endoplasmic reticulum by heterodimerization with GHS-R1b, and constitutive activation of phospholipase C is attenuated due to decreased cell surface expression of ghrelin receptors. However, sufficient ghrelin receptor homodimers can still be expressed on the cell surface for maximal responses to agonist stimulation.

Mechanism of GABAB receptor‐induced BDNF secretion and promotion of GABAA receptor membrane expression

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.

MicroRNA-21 Deficiency Leads to an Attenuated Asthma Response in Mice

M O N D A Y 847 Effects Of Neuropeptide Y On Monocyte-derived Dendritic Cell Function M. Pham, SHAO Z., L. Lorence, R. Gaurav, D. K. Agrawal; Center for Clinical & Translational Science, Creighton University School of Medicine, OMAHA, NE. RATIONALE: Neuropeptide Y (NPY) plays a role in inflammatory disease with current literature demonstrating its modulating effects on macrophages and lymphocytes. However, its effects on dendritic cells are unknown. In this study, the effects of NPYon monocyte-derived dendritic cell (MDDC) functions were examined. METHODS: Monocytes were separated from healthy donor blood. Differentiation into immature MDDCs was achieved with IL-4 and GMCSFwithmaturation induced byTNFa. NPY receptor expressionwas examinedusing qPCRand immunofluorescence.MDDCsMigration in response to NPY was examined in vitro using a TransWell migration assay. The antigen uptake ofMDDCswasmeasured using flow cytometry. NPY receptor antagonists, BIBO3304,BIIE 0246, andNPY5RA972were used to define the role of NPY-Y1R, NPY-Y2R, and NPY-Y5R, respectively. RESULTS: Immature and mature MDDCs express NPYand NPY1, 2, 4, and 5 receptor mRNA transcripts with immunofluorescence confirming NPY1, 2, and 5 receptor membrane expression. During maturation, MDDCs down-regulated expression of the NPY1, 2, and 4 receptor but up-regulated NPY and the NPY5 receptor. Under NPY treatment, mature MDDCs up-regulated NPY1 and NPY5 receptors, whereas immature MDDCs respondedwith little change. NPY treatment increased surface expression of HLA-DR yet decreased surface expression of CCR7, CD83, and CD26 on immature MDDCs. NPY increased migration of mature MDDCs andmay serve as a chemoattractant. Additionally, NPY decreased endocytic activity of immature MDDCs which appears to be mediated by the NPY5 receptor. CONCLUSIONS: NPY modulates dendritic cell function and may contribute to inflammatory disease, at least, in part through dendritic cells.

Channel-Opening Kinetic Mechanism for Human Wild-Type Gluk2 and the M867i Mutant Kainate Receptor

GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the autism mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether this mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 (rGluK2) kainate receptor as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ∼1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminus. Our results also show that the human GluK2 has a ∼3-fold smaller channel-opening rate constant but an identical channel-closing rate constant, thus lowering the channel-opening probability to 0.85 as compared to 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K1for hGluK2 is ∼2-fold lower than rGluK2. Our results therefore suggest that hGluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.

Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Mechanisms underlying purinergic P2X3 receptor-mediated mechanical allodynia induced in diabetic rats.

BackgroundDiabetic neuropathy is a common neuropathy associated with paresthaesia and pain. The mechanisms underlying the painful conditions are not well understood. The aim of this study is to investigate the participation of purinergic P2X3 receptors in painful diabetic neuropathy.ResultsDiabetes was induced by an intraperitoneal injection of streptozotocin (STZ). We showed that mechanical allodynia was induced two weeks after a STZ injection and lasted for at least another seven weeks. The mechanical allodynia was significantly attenuated by peripheral administration of the P2X receptor antagonists, PPADS or TNP-ATP. DiI was subcutaneously injected into the rat hindpaw to label hindpaw-innervated dorsal root ganglion (DRG) neurons. ATP activated fast-inactivating P2X3 receptor-mediated currents in the labeled DRG neurons were studied. ATP responses in STZ-treated rats were ~2-fold larger than those in control rats. Furthermore, the expression of P2X3 receptor proteins in the plasma membrane of L4-6 DRGs of STZ rats was significantly enhanced while the total expression of P2X3 receptors remained unaltered.ConclusionsThese results indicate that a large enhancement of P2X3 receptor activity and an increase in the membrane expression of P2X3 receptors contribute to the development of chronic pain in STZ-induced diabetic rats and suggest a possible target for the treatment of diabetic neuropathic pain.

TNF‐related apoptosis‐inducing ligand cooperates with NSAIDs via activated Wnt signalling in (pre)malignant colon cells

TNF-related apoptosis-inducing ligand (TRAIL) receptor agonistic agents and non-steroidal anti-inflammatory drugs (NSAIDs) are interesting agents for the chemoprevention and treatment of colorectal cancer. We investigated whether NSAIDs sensitize colon cancer and adenoma cell lines and ex vivo cultured human adenomas to recombinant human (rh)TRAIL. Involvement of the crucial Wnt signalling pathway in the sensitization of colon cancer cells was examined. Five colon cancer and two adenoma cell lines, human ex vivo adenomas and normal colonic epithelium were treated with aspirin or sulindac combined with rhTRAIL. Apoptosis levels, expression of intracellular proteins and TRAIL receptor membrane expression were assessed. Ls174T cells stably transfected with an inducible dominant negative TCF-4 (dnTCF-4) construct served to analyse the role of Wnt pathway activation. Both rhTRAIL-sensitive and -resistant colon cancer cell lines were strongly sensitized to rhTRAIL by aspirin (maximum enhancement ratio, 7.1). Remarkably, in adenoma cell lines sulindac enhanced rhTRAIL-induced apoptosis most effectively (maximum enhancement ratio, 2.5). Although membrane TRAIL receptor expression was not affected by NSAIDs, caspase-8 activation was enhanced by combinational treatment. Several proteins from different biological pathways were affected by NSAIDs, indicating complex mechanisms of sensitization. Elimination of TCF-4 completely blocked the sensitizing effect in colon cancer cells. In ex vivo adenomas the combination of sulindac and rhTRAIL increased apoptosis from 18.4% (sulindac) and 17.8% (rhTRAIL) to 28.0% (p = 0.003 and p = 0.005, respectively). It was concluded that NSAID-induced sensitization to rhTRAIL requires TCF-4 activity. Thus, the combination of TRAIL-receptor agonistic agents and NSAIDs is a potentially attractive treatment option for (pre)malignant tumours with constitutively active Wnt signalling, such as colorectal tumours.