Showing papers on "Receptor antagonist published in 2017"

Substance P activates Mas-related G protein–coupled receptors to induce itch

Abstract: Background Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein–coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1. Objective Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs. Methods Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice. Results SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R. Conclusion We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

Dietary and pharmacological treatment of abdominal pain in IBS.

Abstract: This review introduces the principles of visceral sensation and appraises the current approaches to management of visceral pain in functional GI diseases, principally IBS. These approaches include dietary measures including fibre supplementation, low fermentable oligosaccharides, disaccharides, monosaccharides and polyols diet, and pharmacological approaches such as antispasmodics, peppermint oil, antidepressants (tricyclic agents, selective serotonin reuptake inhibitors), 5-HT3 receptor antagonists (alosetron, ondansetron, ramosetron), non-absorbed antibiotic (rifaximin), secretagogues (lubiprostone, linaclotide), μ-opioid receptor (OR) and κ-OR agonist, δ-OR antagonist (eluxadoline), histamine H1 receptor antagonist (ebastine), neurokinin-2 receptor antagonist (ibodutant) and GABAergic agents (gabapentin and pregabalin). Efficacy and safety are discussed based on pivotal trials or published systematic reviews and meta-analysis, expressing ORs or relative risks and their 95% CIs. Potential new approaches may be based on recent insights on mucosal expression of genes, and microRNA and epigenetic markers in human biopsies and in animal models of visceral hypersensitivity.The objectives of this review are to appraise the physiology and anatomy of gut sensation and the efficacy in the relief of visceral pain (typically in IBS) of several classes of therapies. These include fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) and different classes of medications (box 1). Box 1Classes of pharmacological agents for visceral painAntidepressants (tricyclic agents, selective serotonin reuptake inhibitors)Peppermint oil5-HT3 receptor antagonists (alosetron, ondansetron, ramosetron)Non-absorbed antibiotic (rifaximin)Secretagogues (lubiprostone, linaclotide)μ-Opioid receptor (OR) and κ-OR agonist and δ-OR antagonist (eluxadoline)Histamine H1 receptor antagonist (ebastine)Neurokinin-2 receptor antagonist (ibodutant)GABAergic agents (gabapentin and pregabalin).

Targeting the PGD2/CRTH2/DP1 Signaling Pathway in Asthma and Allergic Disease: Current Status and Future Perspectives.

Abstract: Prostaglandin D2 (PGD2) released by degranulating mast cells is believed to play a key role in orchestrating mechanisms of inflammation in allergies and asthma. The biological effects of PGD2 are mediated by D-prostanoid (DP1), CRTH2 (DP2), and thromboxane prostanoid (TP) receptors. The CRTH2 receptor is involved in induction of migration and activation of T helper type 2 (Th2) lymphocytes, eosinophils, and basophils; up-regulation of adhesion molecules; and promotion of pro-inflammatory Th2-type cytokines (interleukin [IL]-4, 5, 13), whereas the DP receptor is associated with relaxation of smooth muscles, vasodilation, inhibition of cell migration, and apoptosis of eosinophils. A number of CRTH2/PGD2 receptor antagonists have been investigated in asthma and allergic diseases. The CRTH2 antagonist (OC000459) or dual CRTH2 and TP receptor antagonist (ramatroban) were effective in reducing eosinophilia, nasal mucosal swelling, and clinical symptoms of allergic rhinitis, with the latter drug registered for clinical use in this indication. OC000459 and setipiprant reduced the late but not early phase of response in an allergen challenge in atopic asthmatics. In persistent asthma, some molecules induced limited improvement in lung function, quality of life, and asthma symptoms (OC000459, BI671800), but in other trials with AMG 853 and AZ1981 these findings were not confirmed. The clear discrepancy between animal studies and clinical efficacy of CRTH2 antagonism in allergic rhinitis, and lack of efficacy in a general cohort of asthmatics, highlight the issue of patient phenotyping. There is no doubt that the PGD2/CATH2/DP1 pathway plays a key role in allergic inflammation and further studies with selective or combined antagonisms in well defined cohorts of patients are needed.

Spatial and temporal boundaries of NMDA receptor hypofunction leading to schizophrenia.

Abstract: The N-methyl-d-aspartate receptor hypofunction is one of the most prevalent models of schizophrenia. For example, healthy subjects treated with uncompetitive N-methyl-d-aspartate receptor antagonists elicit positive, negative, and cognitive-like symptoms of schizophrenia. Patients with anti-N-methyl-d-aspartate receptor encephalitis, which is likely caused by autoantibody-mediated down-regulation of cell surface N-methyl-d-aspartate receptors, often experience psychiatric symptoms similar to schizophrenia initially. However, where and when N-methyl-d-aspartate receptor hypofunction occurs in the brain of schizophrenic patients is poorly understood. Here we review the findings from N-methyl-d-aspartate receptor antagonist and autoantibody models, postmortem studies on N-methyl-d-aspartate receptor subunits, as well as the global and cell-type-specific knockout mouse models of subunit GluN1. We compare various conditional GluN1 knockout mouse strains, focusing on the onset of N-methyl-d-aspartate receptor deletion and on the cortical cell-types. Based on these results, we hypothesize that N-methyl-d-aspartate receptor hypofunction initially occurs in cortical GABAergic neurons during early postnatal development. The resulting GABA neuron maturation deficit may cause reduction of intrinsic excitability and GABA release, leading to disinhibition of pyramidal neurons. The cortical disinhibition in turn could elicit glutamate spillover and subsequent homeostatic down regulation of N-methyl-d-aspartate receptor function in pyramidal neurons in prodromal stage. These two temporally-distinct N-methyl-d-aspartate receptor hypofunctions may be complimentary, as neither alone may not be able to fully explain the entire schizophrenia pathophysiology. Potential underlying mechanisms for N-methyl-d-aspartate receptor hypofunction in cortical GABA neurons are also discussed, based on studies of naturally-occurring N-methyl-d-aspartate receptor antagonists, neuregulin/ErbB4 signaling pathway, and theoretical analysis of excitatory/inhibitory balance.

Medium-chain fatty acids modulate myocardial function via a cardiac odorant receptor.

Abstract: Several studies have demonstrated the expression of odorant receptors (OR) in various human tissues and their involvement in different physiological and pathophysiological processes. However, the functional role of ORs in the human heart is still unclear. Here, we firstly report the functional characterization of an OR in the human heart. Initial next-generation sequencing analysis revealed the OR expression pattern in the adult and fetal human heart and identified the fatty acid-sensing OR51E1 as the most highly expressed OR in both cardiac development stages. An extensive characterization of the OR51E1 ligand profile by luciferase reporter gene activation assay identified 2-ethylhexanoic acid as a receptor antagonist and various structurally related fatty acids as novel OR51E1 ligands, some of which were detected at receptor-activating concentrations in plasma and epicardial adipose tissue. Functional investigation of the endogenous receptor was carried out by Ca2+ imaging of human stem cell-derived cardiomyocytes. Application of OR51E1 ligands induced negative chronotropic effects that depended on activation of the OR. OR51E1 activation also provoked a negative inotropic action in cardiac trabeculae and slice preparations of human explanted ventricles. These findings indicate that OR51E1 may play a role as metabolic regulator of cardiac function.

Melatonin protects against cisplatin-induced ovarian damage in mice via the MT1 receptor and antioxidant activity.

Abstract: This study evaluated the receptor- and/or antioxidant stress-mediated mechanisms by which melatonin prevents the ovarian toxicity of cisplatin treatment. The expression of the MT1 receptor in mouse ovaries was investigated by immunohistochemistry. Pretreatment with melatonin (5, 10, or 20 mg/kg body weight, i.p.) before cisplatin (5 mg/kg body weight, i.p.) was administered to mice once daily for 3 days (phase I). The pharmacological modulation via melatonin type 1 and/or 2 receptors was analyzed by administration of receptor antagonists (luzindole: nonselective MT1/MT2 antagonist; 5 mg/kg body weight or 4-phenyl-2-propionamidotetralin: selective MT2 antagonist; 4mg/kg body weight) once daily for 3 days, 15 min before the treatment with melatonin and cisplatin (phase II). Thereafter, the ovaries were harvested and used for histological (morphology and activation), immunohistochemical (PCNA, activated caspase-3 and bcl-2 expression), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. The expression of the MT1 protein in mouse ovaries was documented. Pretreatment with 20 mg/kg melatonin before cisplatin administration preserved the normal follicular morphology and cell proliferation rate, reduced apoptosis, ROS production, mitochondrial damage and increased GSH expression, as compared to the cisplatin treatment alone. Additionally, administration of the nonselective MT1/MT2 receptor antagonist inhibited the melatonin ovarian protection from the cytotoxic effects of cisplatin. However, administration of a selective MT2 antagonist did not modify the protective effects observed at 20 mg/kg melatonin. In conclusion, pretreatment with 20 mg/kg melatonin effectively protected the ovaries against cisplatin-induced damage. Moreover, the MT1 receptor and melatonin antioxidant effects mediated this cytoprotective activity.Summary SentenceMelatonin attenuated cisplatin-induced ovarian damage in mice, and the MT1 receptor could be used as a promising therapeutic target to the development of novel agents for preserving ovarian function during chemotherapy.

Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies.

Abstract: Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

Synthesis, characterization, and in vitro evaluation of the selective P2Y 2 receptor antagonist AR-C118925

Abstract: The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

P2X7 receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL-1β pathway.

Abstract: Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X7 signal has been shown to activate the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X7 signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin-1β (IL-1β) axis is involved in the process. We explored the relationship between P2X7 receptor (P2X7 R) and IL-1β in the heart tissue of lipopolysaccharide (LPS)-primed naive rats. 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (BzATP), a P2X7 R agonist, induced caspase-1 activation and mature IL-1β release, which was further neutralized by a NLRP3 inhibitor (16673-34-0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X7 R was localized within infiltrated macrophages and observed in parallel with an up-regulation of NLRP3 inflammasome levels and the release of IL-1β in the left ventricle. The administration of A-740003 (a P2X7 R antagonist) significantly prevented the NLRP3/IL-1β increase. A-740003 and/or Anakinra (an IL-1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage-based IL-1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X7 on neural remodelling was mediated at least partially by IL-1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up-regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL-1β. Together, these data indicate that P2X7 R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL-1β axis. Therapeutic interventions targeting P2X7 signal may be a novel approach to ameliorate arrhythmia following MI.

Sigma-1 Receptor Agonists and Their Clinical Implications in Neuropsychiatric Disorders

Abstract: Accumulating evidence suggests that sigma-1 receptors play a role in the pathophysiology of neuropsychiatric diseases, as well as in the mechanisms of some selective serotonin reuptake inhibitors (SSRIs). Among the SSRIs, the order of affinity for sigma-1 receptors is as follows: fluvoxamine > sertraline > fluoxetine > escitalopram > citalopram >> paroxetine. Some SSRIs (e.g., fluvoxamine, fluoxetine and escitalopram) and other drugs (donepezil, ifenprodil, dehydroepiandeterone (DHEA)) potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC12 cells, and these effects could be antagonized by the selective sigma-1 receptor antagonist NE-100. Furthermore, fluvoxamine, donepezil, and DHEA, but not paroxetine or sertraline, improved phencyclidine-induced cognitive deficits in mice, and these effects could be antagonized by NE-100. Several clinical studies showed that sigma-1 receptor agonists such as fluvoxamine and ifenprodil could have beneficial effects in patients with neuropsychiatric disorders. In this chapter, the authors will discuss the role of sigma-1 receptors in the mechanistic action of some SSRIs, donepezil, neurosteroids, and ifenprodil, and the clinical implications for sigma-1 receptor agonists.

Lipopolysaccharide as trigger of platelet aggregation via eicosanoid over-production

Abstract: The effect of lipopolysaccharide (LPS) on platelet aggregation is still controversial. We performed in vitro and ex vivo studies in controls and in patients with community-acquired pneumonia (CAP) to assess the effect of LPS on platelet activation (PA). LPS (15–100 pg/ml) significantly increased PA only if combined with sub-threshold concentrations (STC) of collagen or ADP; this effect was associated with increased platelet H2O2 production, Nox2 activation, PLA2 phosphorylation, thromboxane (Tx)A2 and 8-iso-PGF2α-III, and was inhibited by aspirin, TxA2 receptor antagonist or by Toll-like receptor 4 blocking peptide (TLR4bp). Analysis of up-stream signalling potentially responsible for Nox2 and PLA2 activation demonstrated that LPS-mediated PA was associated with phosphorylation of AKT, p38 and p47phox translocation. In 10 consecutive CAP patients serum endotoxins were significantly higher compared to 10 controls (145 [115–187] vs 18 [6–21] pg/ml; p Supplementary Material to this article is available online at www.thrombosis-online.com

Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells.

Abstract: The β2 adrenergic receptor (β2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following β2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β2AR activation. Consistent with this mechanism, β2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the β2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that β2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.

Enhanced delivery of IL-1 receptor antagonist to the central nervous system as a novel anti-transferrin receptor-IL-1RA fusion reverses neuropathic mechanical hypersensitivity.

Abstract: Neuropathic pain is a major unmet medical need, with only 30% to 35% of patients responding to the current standard of care. The discovery and development of novel therapeutics to address this unmet need have been hampered by poor target engagement, the selectivity of novel molecules, and limited access to the relevant compartments. Biological therapeutics, either monoclonal antibodies (mAbs) or peptides, offer a solution to the challenge of specificity as the intrinsic selectivity of these kinds of molecules is significantly higher than traditional medicinal chemistry-derived approaches. The interleukin-1 receptor system within the spinal cord has been implicated in the amplification of pain signals, and its central antagonism provides relief of neuropathic pain. Targeting the IL-1 system in the spinal cord with biological drugs, however, raises the even greater challenge of delivery to the central compartment. Targeting the transferrin receptor with monoclonal antibodies has proved successful in traversing the endothelial cell-derived blood-brain barrier and delivering proteins to the central nervous system. In this study, we describe a novel construct exemplifying an engineered solution to overcome these challenges. We have generated a novel anti-transferrin receptor-interleukin-1 receptor antagonist fusion that transports to the central nervous system and delivers efficacy in a model of nerve ligation-induced hypersensitivity. Approaches such as these provide promise for novel and selective analgesics that target the central compartment.

Ketamine Increases the Function of γ-Aminobutyric Acid Type A Receptors in Hippocampal and Cortical Neurons.

Abstract: Background:The “dissociative ” general anesthetic ketamine is a well-known N-methyl-D-aspartate receptor antagonist. However, whether ketamine, at clinically relevant concentrations, increases the activity of inhibitory γ-aminobutyric acid (GABA) receptor type A (GABAA) receptors in different brain

Inhibition of A2A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455.

Abstract: Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1, A2A, A2B, and A3 receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N5-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A2AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A2AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A2AAR agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A2AAR antagonist TP455, as well as by the reference A2AAR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A2AAR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by A2AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the A2AAR. In conclusion, the A2AAR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective A2AAR antagonists as potential new therapeutics.

The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1.

Abstract: The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated in vitro. The acute glucose lowering effect was studied in lean wild-type and GPR120 null mice following oral or intravenous glucose tolerance tests. In vitro, in GPR120 overexpressing cells, both agonists signaled through Gαq, Gαs and the β-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both in vitro in STC-1 cells and in vivo following oral administration. In vivo GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 null mice. Exendin 9–39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

Abstract: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Role of microglial amylin receptors in mediating beta amyloid (Aβ)-induced inflammation.

Abstract: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer’s disease (AD) pathology. Amyloid beta (Aβ) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aβ activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aβ1–42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. Acute applications of human amylin or Aβ1–42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1β, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1β. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. Microglial amylin receptors mediate Aβ-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Comparative Effects of LY3020371, a Potent and Selective Metabotropic Glutamate (mGlu) 2/3 Receptor Antagonist, and Ketamine, a Noncompetitive N-Methyl-d-Aspartate Receptor Antagonist in Rodents: Evidence Supporting the Use of mGlu2/3 Antagonists, for the Treatment of Depression.

Abstract: The ability of the N-methyl-d-aspartate receptor antagonist ketamine to alleviate symptoms in patients suffering from treatment-resistant depression (TRD) is well documented. In this paper, we directly compare in vivo biologic responses in rodents elicited by a recently discovered metabotropic glutamate (mGlu) 2/3 receptor antagonist 2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY3020371) with those produced by ketamine. Both LY3020371 and ketamine increased the number of spontaneously active dopamine cells in the ventral tegmental area of anesthetized rats, increased O2 in the anterior cingulate cortex, promoted wakefulness, enhanced the efflux of biogenic amines in the prefrontal cortex, and produced antidepressant-related behavioral effects in rodent models. The ability of LY3020371 to produce antidepressant-like effects in the forced-swim assay in rats was associated with cerebrospinal fluid (CSF) drug levels that matched concentrations required for functional antagonist activity in native rat brain tissue preparations. Metabolomic pathway analyses from analytes recovered from rat CSF and hippocampus demonstrated that both LY3020371 and ketamine activated common pathways involving GRIA2 and ADORA1. A diester analog of LY3020371 [bis(((isopropoxycarbonyl)oxy)-methyl) (1S,2R,3S,4S,5R,6R)-2-amino-3-(((3,4-difluorophenyl)thio)methyl)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylate (LY3027788)] was an effective oral prodrug; when given orally, it recapitulated effects of intravenous doses of LY3020371 in the forced-swim and wake-promotion assays, and augmented the antidepressant-like effects of fluoxetine or citalopram without altering plasma or brain levels of these compounds. The broad overlap of biologic responses produced by LY3020371 and ketamine supports the hypothesis that mGlu2/3 receptor blockade might be a novel therapeutic approach for the treatment of TRD patients. LY3020371 and LY3027788 represent molecules that are ready for clinical tests of this hypothesis.

The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

Abstract: Background/aims In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway.

A new serotonin 5-HT 6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding

Abstract: Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer’s disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

Response to Interleukin (IL)-17 Inhibition in an Adolescent With Severe Manifestations of IL-36 Receptor Antagonist Deficiency (DITRA).

Abstract: Response to Interleukin (IL)-17 Inhibition in an Adolescent With Severe Manifestations of IL-36 Receptor Antagonist Deficiency (DITRA) Deficiency of the interleukin (IL)-36 receptor antagonist (DITRA) is a recently described autosomal recessive auto-inflammatory syndrome caused by mutations in the IL36RN gene, characterized clinically by recurrent episodes of generalized skin pustulation, fever, systemic inflammation and leukocytosis.1,2 A uniformly effective treatment for DITRA has not been identified.

Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2-USP5 interactions in the pain pathway.

Abstract: We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1–expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers.

The P2X7 receptor links mechanical strain to cytokine IL-6 up-regulation and release in neurons and astrocytes.

Abstract: Background Mechanical strain in neural tissues can lead to the upregulation and release of multiple cytokines including IL-6. In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Methods Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 hrs. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist BzATP were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice. Results Acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1m, IL24, Tnf, Csf1 and Lif also increased more than two-fold, while Tnfsf11, Gdf9 and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. Conclusions The mechanosensitive upregulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response. This article is protected by copyright. All rights reserved.

Development of a human vasopressin V 1a -receptor antagonist from an evolutionary-related insect neuropeptide

Abstract: Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

Evaluation of JNJ-54717793 a Novel Brain Penetrant Selective Orexin 1 Receptor Antagonist in Two Rat Models of Panic Attack Provocation.

Abstract: Orexin neurons originating in the perifornical and lateral hypothalamic area are highly reactive to anxiogenic stimuli and have strong projections to anxiety and panic-associated circuitry. Recent studies support a role for the orexin system and in particular the orexin 1 receptor (OX1R) in coordinating an integrative stress response. However, no selective OX1R antagonist has been systematically tested in two preclinical models of using panicogenic stimuli that induce panic attack in the majority of people with panic disorder, namely an acute hypercapnia-panic provocation model and a model involving chronic inhibition of GABA synthesis in the perifornical hypothalamic area followed by intravenous sodium lactate infusion. Here we report on a novel brain penetrant, selective and high affinity OX1R antagonist JNJ-54717793 (1S,2R,4R)-7-([(3-fluoro-2-pyrimidin-2-ylphenyl)carbonyl]-N-[5-(trifluoromethyl)pyrazin-2-yl]-7-azabicyclo[2.2.1]heptan-2-amine). JNJ-54717793 is a high affinity/potent OX1R antagonist and has an excellent selectivity profile including 50 fold versus the OX2R. Ex vivo receptor binding studies demonstrated that after oral administration JNJ-54717793 crossed the blood brain barrier and occupied OX1Rs in the rat brain. While JNJ-54717793 had minimal effect on spontaneous sleep in rats and in wild-type mice, its administration in OX2R knockout mice, selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. JNJ-54717793 attenuated CO2 and sodium lactate induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. These data confirm that selective OX1R antagonism may represent a novel approach of treating anxiety disorders, with no apparent sedative effects.