A multicenter audit of the use of bronchoscopy during open and thoracoscopic repair of esophageal atresia with tracheoesophageal fistula.

BACKGROUND: Esophageal atresia (EA) with tracheoesophageal fistula is usually repaired in the neonatal period. Preferential ventilation through the fistula can lead to gastric distension. Bronchoscopy has a role in defining the site and size of the fistula, and may be carried out by the surgeon or the anesthetist. The use of bronchoscopy varies across different institutions. METHODS: This is a multicenter case note review of infants with EA with tracheoesophageal fistula who underwent surgery between January 2010 and December 2015. This retrospective audit aims primarily to document the use of bronchoscopy during open and thoracoscopic repair at a selection of United Kingdom centers. Respiratory complications, that is relating to airway management, the respiratory system, and difficulty with ventilation, at induction and during surgery, are recorded. The range of techniques for anesthesia and analgesia in these centers is noted. RESULTS: Bronchoscopy was carried out in 52% of cases. The incidence of respiratory complications was 7% at induction and 21% during surgery. Thoracoscopic repair usually took longer. One center used high-frequency oscillatory ventilation, on an elective basis during thoracoscopic repair, to facilitate surgical access and address concerns about hypoxemia and hypercarbia. CONCLUSION: The use of bronchoscopy varies considerably between institutions. Infants undergoing tracheoesophageal fistula repair are at risk of perioperative respiratory morbidity. The advent of thoracoscopic repair has introduced further variation.

The Role of Metabotropic Glutamate Receptor 1 Dependent Signaling in Glioma Viability.

Glioma refers to malignant central nervous system tumors that have histologic characteristics in common with glial cells. The most prevalent type, glioblastoma multiforme, is associated with a poor prognosis and few treatment options. On the basis of reports of aberrant expression of mGluR1 mRNA in glioma, evidence that melanoma growth is directly influenced by glutamate metabotropic receptor 1 (mGluR1), and characterization of ß-arrestin-dependent prosurvival signaling by this receptor, this study investigated the hypothesis that glioma cell lines aberrantly express mGluR1 and depend on mGluR1-mediated signaling to maintain viability and proliferation. Three glioma cell lines (Hs683, A172, and U87) were tested to confirm mGluR1 mRNA expression and the dependence of glioma cell viability on glutamate. Pharmacologic and genetic evidence is presented that suggests mGluR1 signaling specifically supports glioma proliferation and viability. For example, selective noncompetitive antagonists of mGluR1, CPCCOEt and JNJ16259685, decreased the viability of these cells in a dose-dependent manner, and glutamate metabotropic receptor 1 gene silencing significantly reduced glioma cell proliferation. Also, results of an anchorage-independent growth assay suggested that noncompetitive antagonism of mGluR1 may decrease the tumorigenic potential of Hs683 glioma cells. Finally, data are provided that support the hypothesis that a ß-arrestin-dependent signaling cascade may be involved in glutamate-stimulated viability in glioma cells and that ligand bias may exist at mGluR1 expressed in these cells. Taken together, the results strongly suggest that mGluR1 may act as a proto-oncogene in glioma and be a viable drug target in glioma treatment.

Experiências de uma ONG na promoção de direitos e saúde trans em São Paulo / Experiences of a NGO with promoting trans rights and health care in São Paulo

Este artigo procura expor as ações realizadas pelo Instituto Cultural Barong junto à transexuais, visando à promoção de seus direitos, incluindo o de saúde e prevenção de IST/HIV. São relatadas as estratégias de promoção de informações por meio do desenvolvimento de materiais educativos, exposições, ações de campo e inclusive cursos produzidos para este público, procurando atender suas necessidades, locais de circulação de trabalho ou sociabilidade, linguagem e demandas. As estratégias foram consideradas de sucesso pela sua adesão e receptividade comprovada no público e ambientes integrados.

The (α4)3(ß2)2 Stoichiometry of the Nicotinic Acetylcholine Receptor Predominates in the Rat Motor Cortex.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(ß2)3 and (α4)3(ß2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and ß2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4ß2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or ß2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/ß2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4ß2 receptors in this brain region. We report that the majority of α4ß2 nAChRs in this brain region possess a stoichiometry of the (α4)3(ß2)2 LS subtype.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Rat α3ß4 Nicotinic Cholinergic Receptors.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3ß4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3ß4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3ß4 and α4ß2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3ß4 receptors over α4ß2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3ß4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3ß4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat α3ß4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4ß2 nAChRs. Both α3ß4 and α4ß2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3ß4 receptors than rat α3ß4 and human α4ß2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3ß4 nAChR and shortest for the human α4ß2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors.

The elemental anion chloride is generally considered a passive participant in neuronal excitability, and has never been shown to function as an agonist in its own right. We show that the antagonist-mediated, glutamate-independent inverse agonism of group II and III metabotropic glutamate (mGlu) receptors results from inhibition of chloride-mediated activation. In silico molecular modeling, site-directed mutagenesis, and functional assays demonstrate (1) that chloride is an agonist of mGlu3, mGlu4, mGlu6, and mGlu8 receptors with its own orthosteric site, and (2) that chloride is not an agonist of mGlu2 receptors. Molecular modeling-predicted and site-directed mutagenesis supported that this unique property of mGlu2 receptors results from a single divergent amino acid, highlighting a molecular switch for chloride insensitivity that is transduced through an arginine flip. Ultimately, these results suggest that activation of group II and III mGlu receptors is mediated not only by glutamate, but also by physiologically relevant concentrations of chloride.

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

From student to steward: the Interdisciplinary Program in Neuroscience at Georgetown University as a case study in professional development during doctoral training.

A key facet of professional development is the formation of professional identity. At its most basic level, professional identity for a scientist centers on mastery of a discipline and the development of research skills during doctoral training. To develop a broader understanding of professional identity in the context of doctoral training, the Carnegie Initiative on the Doctorate (CID) ran a multi-institutional study from 2001 to 2005. A key outcome of the CID was the development of the concept of 'stewards of the discipline'. The Interdisciplinary Program in Neuroscience (IPN) at Georgetown University participated in CID from 2003 to 2005. Here, we describe the IPN and highlight the programmatic developments resulting from participation in the CID. In particular, we emphasize programmatic activities that are designed to promote professional skills in parallel with scientific development. We describe activities in the domains of leadership, communication, teaching, public outreach, ethics, collaboration, and mentorship. Finally, we provide data that demonstrate that traditional metrics of academic success are not adversely affected by the inclusion of professional development activities in the curricula. By incorporating these seven 'professional development' activities into the required coursework and dissertation research experience, the IPN motivates students to become stewards of the discipline.

A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

Clinical process in an integrative psychotherapy for self-wounds.

In this article, I will briefly describe the clinical process of an integrative psychotherapy for the healing of self-wounds, including its intended interventions and the variability of their application and outcome. Four specific strategies will be considered, including (a) the role of empathy throughout the course of therapy; (b) exposure therapy as a paradigmatic treatment for the treatment of feared thoughts, behavior, and emotions; (c) focusing and other experiential interventions for eliciting self-wounds; and (d) modification and healing of self-wounds with an individualized array of psychodynamic, experiential, and cognitive-behavioral strategies. In addition, we will briefly consider the impact of transference and countertransference on the trajectory of therapy.

Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain.

Chronic nicotine administration increases α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4ß2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ∼150 times its affinity for α4ß2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4ß2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.

Bridging the gap between clinical research and clinical practice: introduction to the special section.

This Special Section, developed by the American Psychology Association's Division 12 (Clinical) 2011 Committee on Science and Practice, highlights different ideas to help bridge the gap between clinical research and clinical practice, and notes recent innovations that help make research-practice integration feasible. The articles consider how to break down the barriers to enhance researcher-practitioner dialogue, as well as how to make ongoing outcome assessment feasible for clinicians. Moreover, the articles address how to promote training in evidence-based practice, and how to translate efficacy research into clinical practice and clinical insight into empirical study to better establish a two-way bridge between research and practice. Ultimately, we hope this series can speak to many different types of psychologists, whether they work mainly as researchers or practitioners, so they can see new ways to integrate and learn from both research and practice.

Healing the research-practice split: let's start with me.

This article highlights many of the major issues that separate the researcher from the clinician and then suggests some possible solutions. The format involves an enacted two-chair dialogue between my researcher side and my therapist side. The dialogue involves three phases. Phase 1 involves each side presenting a straw man of the other's position. In Phase 2, the two sides engage in a creative dialogue regarding many of the issues that divide them. Phase 3 eventuates in a series of synthetic solutions that honors the concerns and core values of each side. The solutions include: (a) broadening the definition of clinically useful research, (b) researcher-practitioner collaboration, (c) appropriate dissemination of user-friendly research findings, (d) translating research findings into experience-near procedures such as the systematic case studies, and (e) providing clinicians with easy access to a video archive of research therapies.

A translational model of research-practice integration.

We propose a four-level, recursive Research-Practice Integration framework as a heuristic to (a) integrate and reflect on the articles in this Special Section as contributing to a bidirectional bridge between research and practice, and (b) consider additional opportunities to address the research-practice gap. Level 1 addresses Treatment Validation studies and includes an article by Lochman and colleagues concerning the programmatic adaptation, implementation, and dissemination of the empirically supported Coping Power treatment program for youth aggression. Level 2 translation, Training in Evidence-Based Practice, includes a paper by Hershenberg, Drabick, and Vivian, which focuses on the critical role that predoctoral training plays in bridging the research-practice gap. Level 3 addresses the Assessment of Clinical Utility and Feedback to Research aspects of translation. The articles by Lambert and Youn, Kraus, and Castonguay illustrate the use of commercial outcome packages that enable psychotherapists to integrate ongoing client assessment, thus enhancing the effectiveness of treatment implementation and providing data that can be fed back to researchers. Lastly, Level 4 translation, the Cross-Level Integrative Research and Communication, concerns research efforts that integrate data from clinical practice and all other levels of translation, as well as communication efforts among all stakeholders, such as researchers, psychotherapists, and clients. Using a two-chair technique as a framework for his discussion, Wolfe's article depicts the struggle inherent in research-practice integration efforts and proposes a rapprochement that highlights advancements in the field.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Morris B. Parloff (1918-2011).

Morris B. Parloff was considered an elder statesman in the field of psychotherapy research, and his wisdom and stewardship were of enormous benefit to several generations of psychotherapy researchers. Parloff received his bachelor's degree from Western Reserve University (1940), a degree in psychiatric social work from the University of Chicago (1942), and a doctorate in psychology from Western Reserve University (1953). Parloff spent most of his career (from 1953 until his retirement in 1983) as a researcher and administrator at the National Institute of Mental Health (NIMH). He was also on the senior faculty of the Washington School of Psychiatry, taught or consulted at a number of universities, and maintained a private practice of psychotherapy. In the NIMH intramural program, Parloff was chief of the Section on Personality in the Laboratory of Psychology. There he conducted a major longitudinal study on the personality characteristics of adolescent and adult scientists. In the NIMH extramural program, he served as chief of the Psychotherapy and Behavioral Intervention Section in the Clinical Research Branch (1972-1980), after which he became chief of the new Psychosocial Treatments Research Branch. Throughout his career, from his pioneering teaching and research on group psychotherapy through his 30 years at NIMH, Parloff provided researchers and practitioners with a broad understanding of the field of psychotherapy research, the complexity of its subject matter, and its relationship to the "real world." Often ahead of others in the field, Parloff dealt with many topics that retain their importance today, including the need to carefully define criteria for improvement in psychotherapy, the transmission of values in psychotherapy, the concepts of the placebo and of common factors in psychotherapy research, and the role of the patient-therapist relationship (in both individual and group therapy) and its impact on the outcome of therapy. Starting with a 1979 article in the American Psychologist ("Can Psychotherapy Research Guide the Policymaker? Vol. 34, pp. 296-306), Parloff wrote extensively about the relationship among practitioners, psychotherapy researchers, and policymakers. To promote the systematic use of clinical trials and address methodological issues in the field, Parloff obtained support to fund the first NIMH multisite collaborative outcome study in the field of psychotherapy. He and the first author (I. E.) then designed and launched the NIMH Treatment of Depression Collaborative Research Program. This study would serve as a model for future collaborative research by independent investigators. (PsycINFO Database Record (c) 2012 APA, all rights reserved).

Endogenously expressed muscarinic receptors in HEK293 cells augment up-regulation of stably expressed α4ß2 nicotinic receptors.

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4ß2 nAChR binding site density and ß2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4ß2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and ß2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and ß2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons.

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [¹²5I]epibatidine ([¹²5I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, ß2 and α5 subunits, but α2, α3, α6 and ß4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the ß2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.