Nicotine stimulates ion transport via metabotropic β4 subunit containing nicotinic ACh receptors

Background and Purpose Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown. Experimental Approach Transepithelial short circuit currents (ISC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+]i were studied on freshly dissociated mouse tracheal epithelial cells. Key Results Apical application of the nAChR agonist nicotine transiently increased ISC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. α‐Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A‐85380 (α4β2 and α3β4) increased ISC. The antagonists dihydro‐β‐erythroidine (α4β2, α3β2, α4β4 and α3β4), α‐conotoxin MII (α3β2) and α‐conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine‐ and epibatidine‐induced currents were unaltered in β2−/−mice, but in β4−/− mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+‐ATPase inhibitor) or the ryanodine receptor antagonists JTV‐519 and dantrolene there was a reduction in the nicotine‐effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H‐89 and the TMEM16A (Ca2+‐activated chloride channel) inhibitor T16Ainh‐A01 significantly reduced the nicotine‐effect. Conclusion and Implications α3β4 nAChRs are responsible for the nicotine‐induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo. © 2016 Mandl et al

Accurate Prediction of the Response of Freshwater Fish to a Mixture of Estrogenic Chemicals

Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures. We investigated the implications of this by analyzing the combined effects of a multicomponent mixture of five estrogenic chemicals using vitellogenin induction in male fathead minnows as an end point. The mixture consisted of estradiol, ethynylestradiol, nonylphenol, octylphenol, and bisphenol A. We determined concentration–response curves for each of the chemicals individually. The chemicals were then combined at equipotent concentrations and the mixture tested using fixed-ratio design. The effects of the mixture were compared with those predicted by the model of concentration addition using biomathematical methods, which revealed that there was no deviation between the observed and predicted effects of the mixture. These findings demonstrate that estrogenic chemicals have the capacity to act together in an additive manner and that their combined effects can be accurately predicted by concentration addition. We also explored the potential for mixture effects at low concentrations by exposing the fish to each chemical at one-fifth of its median effective concentration (EC(50)). Individually, the chemicals did not induce a significant response, although their combined effects were consistent with the predictions of concentration addition. This demonstrates the potential for estrogenic chemicals to act additively at environmentally relevant concentrations. These findings highlight the potential for existing environmental risk assessment procedures to underestimate the hazard posed by mixtures of chemicals that act via a similar mode of action, thereby leading to erroneous conclusions of absence of risk

Mixtures of Estrogenic Chemicals Enhance Vitellogenic Response in Sea Bass

BACKGROUND: The potential impact of natural and synthetic estrogens on aquatic ecosystems has attracted considerable attention because it is currently accepted that their joint effects are more severe when they are present in mixtures. Although it is well-known that they occur as mixtures in the marine environment, there is little information about the combined effects of estrogenic chemicals on marine biota. OBJECTIVE: In 14-day tests with juvenile sea bass, we analyzed singly and in combination the estrogenic activity of estradiol (E2), ethynylestradiol (EE2), and bisphenol A (BPA) using vitellogenin induction as an end point. METHODS: Fish were exposed to each compound, and on the basis of these concentration-response data, we predicted mixture effects by applying the model of concentration addition. The mixtures were tested using a fixed-ratio design, and the resulting mixture effects were compared to the predictions. RESULTS: EE2 was the most potent steroid, with an EC50 (median effective concentration) of 0.029 μg/L, 3.6 times more potent than E2 (EC50 = 0.104 μg/L); BPA was the least potent chemical, with an EC50 of 77.94 μg/L. The comparative assessment yielded a good agreement between observed and predicted mixture effects. CONCLUSIONS: This study demonstrates the potential hazard of these compounds to seawater life by their ability to act together in an additive manner. It provides evidence that concentration addition can be used as a predictive tool for assessing the combined effects of estrogenic chemicals in marine ecosystems

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABA(A) receptors containing the alpha 5 subunit

We have synthesized and characterized MP-III-022 ((R)-8-ethynyl 6 (2 fluorophenyl)-N,4-dimethyl4H-benzo[f]imidazo[1,5-alpha][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy selective positive allosteric modulator of GABA(A) receptors containing the alpha 5 subunit (alpha 5GABA(A)Rs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 110 mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of alpha 5 beta gamma 2 GABA(A) receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2'F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200 mg/kg), but at the price of activating non-alpha 5 GABA(A)Rs as well as the desired alpha 5GABA(A)Rs at the highest dose. At the dose of 10 mg/kg, which elicits a strong positive modulation of alpha 5GABA(A)Rs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10 mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of alpha 5GABA(A)Rs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism

Detection Methods for Autoantibodies in Suspected Autoimmune Encephalitis

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis

Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of alpha 6 GABA(A) receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors containing the alpha 6 subunit are located in trigeminal ganglia, and their reduction by small interfering RNA increases inflammatory temporomandibular and myofascial pain in rats. We thus hypothesized that enhancing their activity may help in neuropathic syndromes originating from the trigeminal system. Here, we performed a detailed electrophysiological and pharmacokinetic analysis of two recently developed deuterated structurally similar pyrazoloquinolinone compounds. DK-I-56-1 at concentrations below 1 mu M enhanced gamma-aminobutyric acid (GABA) currents at recombinant rat alpha 6 beta 3 gamma 2, alpha 6 beta 3 delta and alpha 6 beta 3 receptors, whereas it was inactive at most GABA(A) receptor subtypes containing other alpha subunits. DK-I-87-1 at concentrations below 1 mu M was inactive at alpha 6-containing receptors and only weakly modulated other GABA(A) receptors investigated. Both plasma and brain tissue kinetics of DK-I-56-1 were relatively slow, with half-lives of 6 and 13 hr, respectively, enabling the persistence of estimated free brain concentrations in the range 10-300 nM throughout a 24-hr period. Results obtained in two protocols of chronic constriction injury of the infraorbital nerve in rats dosed intraperitoneally with DK-I-56-1 during 14 days after surgery or with DK-I-56-1 or DK-I-87-1 during 14 days after trigeminal neuropathy were already established, demonstrated that DK-I-56-1 but not DK-I-87-1 significantly reduced the hypersensitivity response to von Frey filaments. Significance Neuropathic pain induced by trigeminal nerve damage is poorly controlled by current treatments. DK-I-56-1 that positively modulates alpha 6 GABA(A) receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo. © 2016 Mandl et al

Project GRACE: A grid based search tool for the global digital library

The paper will report on the progress of an ongoing EU project called GRACE - Grid Search and Categorization Engine (http://www.grace-ist.org). The project participants are CERN, Sheffield Hallam University, Stockholm University, Stuttgart University, GL 2006 and Telecom Italia. The project started in 2002 and will finish in 2005, resulting in a Grid based search engine that will search across a variety of content sources including a number of electronic thesis and dissertation repositories. The Open Archives Initiative (OAI) is expanding and is clearly an interesting movement for a community advocating open access to ETD. However, the OAI approach alone may not be sufficiently scalable to achieve a truly global ETD Digital Library. Many universities simply offer their collections to the world via their local web services without being part of any federated system for archiving and even those dissertations that are provided with OAI compliant metadata will not necessarily be picked up by a centralized OAI Service Provider as the collection might not be officially registered as an OAI data provider. GRACE is an attempt to apply an innovative Grid-based solution that will meet the challenges of searching a global heterogeneous collection of documents. The goal of the project is to build a distributed search and categorization engine that will run on the European Data Grid (EDG) and its successor, the Enabling Grids for E-science in Europe (EGEE). The main difference between GRACE and existing search engines is that GRACE has no centralized index. Instead, it will rely on local indexes or search interfaces that are dispersed across web services around the world. These local sources can use different protocols including http, OAI-PMH and Z39.50. In order to include and index even document collections offering no local search possibilities at all, GRACE will use a native search engine based on Lucene. This decentralized approach, along with the scalable processing power provided by the Grid will result in the following advantages to users: Advanced search capabilities which are flexible enough to allow the broadest possible features given the content sources selected for searching. Increased currency of information and indexes. On-the-fly categorization of documents: the search engine will be capable of dynamically categorizing documents but will also work with existing meta-data and thesauri when desired. Multiple languages for searching and result presentation (starting with English, Italian, Swedish and German). Both anonymous and registered users. Collaboration: Documents or collections shared by registered users or groups. The list of contents sources that the GRACE engine will search is still being developed, but thesis collections in Germany, Sweden and Switzerland are already included. The list will be expanded to include other sources as soon as the tool is up and running. This paper will be a description of the search tool as well as an invitation to collaboration