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Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [3H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Glucocorticoids affect cellular and molecular events in brains by modulating the expression of many genes during stress. In the present study, we examined the regulatory effect of corticosterone on the expression and function of the norepinephrine transporter (NET) in vitro. The results show that exposure of SK-N-BE(2)C cells to corticosterone for 14 days significantly increased mRNA (up to 43%) and protein (up to 71%) levels of NET in the concentration- dependent manner. Longer exposure (21 days) resulted in greater increases in the levels of mRNAs (up to about 160%) and proteins (up to about 250%) of the NET. The up-regulatory effect of corticosterone on NET expression lasted a persistent period after cessation of exposure. Associated with the corticosterone-induced enhancement in NET expression, there was a parallel increase in the uptake of [3H]norepinephrine by SK-N-BE(2)C cells. Increased NET expression and function were abolished after exposure of cells to corticosterone in combination with mifepristone or spironolactone, two specific antagonists of corticosteroid receptors. This is consistent with the hypothesis that corticosterone-induced NET up-regulation is mediated by corticosteroid receptors. Nevertheless, there was no synergistic effect for a combination of both corticosteroid receptor antagonists. A similar up-regulation of NET protein levels was also observed after exposing PC12 cells to corticosterone. The present findings demonstrate that corticosterone up-regulates the expression and function of NET in vitro, indicating the action of corticosterone on the noradrenergic phenotype may play an important role in the correlation between stress and the development of depression.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

La schizophrénie est une pathologie sévère et fréquente. Elle constitue un problème majeur de santé publique. Les traitements disponibles présentent des problèmes de tolérance non négligeables et leur efficacité reste modérée. La recherche en pharmacogénétique des antipsychotiques a pour objectif d’aider les prescripteurs à choisir les traitements de façon plus rationnelle. Les carences méthodologiques des premières études réalisées expliquent sans doute le peu de résultat répliqué à ce jour. Dans une cohorte de patients schizophrènes caucasiens traités par olanzapine ou rispéridone et évaluée prospectivement pour l’efficacité et la tolérance du traitement, nous avons d’abord recherché des critères cliniques permettant de prédire la réponse au traitement. L’age précoce de début des troubles et la durée de la maladie sont des prédicteurs individuels de la mauvaise réponse au traitement. Nous avons également étudié l’implication de variants génétiques du transporteur de la noradrénaline dans l’efficacité des traitements. Nous avons observé l’implication de deux polymorphismes dans la décroissance des symptômes positifs sous traitement. L’analyse de l’impact du variant C825T de la GNB3 dans la prise de poids sous antipsychotique n’a pas retrouvé d’association significative. Enfin, nous avons étudié l’ensemble des variants alléliques du cytochrome P450 2D6 dans cette cohorte de patients schizophrènes comparée à des témoins. L’allèle CYP2D6*2 était associé à un effet protecteur vis à vis de la schizophrénie. Les associations retrouvées devraient aider à mieux comprendre les mécanismes physiopathologiques impliqués dans la schizophrénie et la réponse au traitement / Schizophrenia is a frequent and severe disease. It constitutes a major public health problem. All the available treatments however, have significant adverse side-effects and their efficacy remains moderate. The aim of pharmacogenetic research is to help practitioners to choose treatments in a more rational way. The methodological limits of the first published studies probably explain the lack of replication of such studies. In a prospective study of a sample of Caucasian schizophrenic patients treated with olanzapine or risperidone, clinical criteria were assessed as factors that may predict drug response. Early onset and duration of the disease, individually predicted an unfavourable drug response. We also studied genetic variants of the norepinephrine transporter to see how they may affect antipsychotic drug efficacy. Two polymorphisms were associated with a reduction in positive symptoms in treated schizophrenic patients. No association between the GBN3 C825T variant and weight gain in patients treated by antispychotic drugs was observed. Finally, we genotyped all the cytochrome P450 2D6 allelic variants in the same Caucasian schizophrenic sample and a Caucasian origin control cohort. The CYP2D6*2 allele was strongly associated with protection towards schizophrenia. The two observed associations may help to better understand the still unwell known physiopathological mechanisms implicated in schizophrenia aetiology and antipsychotic drug response

Hintergrund. Untersuchungen bei Patienten mit funktionellen Mutationen des Noradrenalin-Transporter-Gens deuten darauf hin, dass eine verminderte Noradrenalin-Wiederaufnahme zur Pathophysiologie der idiopathischen Orthostatischen Intoleranz beitragen kann. Methoden. In einer doppeltblinden, placebokontrollierten, randomisierten Studie wurden die kardiovaskulären Effekte einer selektiven pharmakologischen Noradrenalin-Wiederaufnahmehemmung (2x 8 mg Reboxetin p.o.) bei gesunden Testpersonen untersucht. In der ersten Teilstudie (n=23) wurden autonome Funktionstests und eine Kipptischuntersuchung durchgeführt. Herzfrequenz, Blutdruck und zerebrale Blutflussgeschwindigkeit wurden dabei kontinuierlich nicht-invasiv gemessen. Herzzeitvolumen und peripherer Widerstand wurden impedanzkardiografisch bestimmt. In einer zweiten Teilstudie (n=10) wurde der Effekt akuter selektiver Noradrenalin-Wiederaufnahmehemmung auf die Sensitivität gegenüber blutdruckwirksamer Pharmaka getestet. Dafür wurden steigende intravenöse Dosen von Phenylephrin, Isoproterenol und Nitroprussid gegeben und die Änderungen von Blutdruck und Herzfrequenz in individuellen Dosis-Wirkungskurven bestimmt. Ergebnisse. Unter Noradrenalin-Wiederaufnahmehemmung waren Herzfrequenz und Blutdruck im Liegen erhöht. Während der Kipptischuntersuchung führte die Noradrenalin-Wiederaufnahmehemmung zu einer deutlich gesteigerten Herzfrequenz (120+/-3 /min vs. 83+/-3 /min unter Placebo, p / Background. Observations in patients with functional mutations of the norepinephrine transporter (NET) gene suggest that impaired norepinephrine uptake may contribute to idiopathic orthostatic intolerance. Methods. In a double-blind, placebo-controlled, and randomized crossover study we investigated the cardiovascular effects of acute selective pharmacological inhibition of the norepinephrine transporter (2x 8mg reboxetine p.o.) on healthy volunteers. Autonomic function test and a graded head-up tilt test were performed (n=23). Heart rate, blood pressure and cerebral blood flow velocity were measured non-invasively and continuously. Cardiac output and peripheral resistance were measured by impedance cardiography. In a smaller subset of volunteers (n=10) we also investigated the effects of acute inhibition of the Norepinephrine transporter on the sensitivity to pressor and depressor agents. Therefore, increasing doses of phenylephrine, isoproterenol, and nitroprusside were administered intravenously and individual dose-response-curves were plotted. Results. Norepinephrine reuptake inhibition increased both, supine heart rate and blood pressure. During head-up tilt, heart rate was profoundly increased with norepinephrine reuptake inhibition (120+/-3 /min vs. 83+/-3 /min with placebo, p

Práca sa zaoberá in silico dizajnom a validáciou peptidov založených na konotoxíne - MrIA, izolovaného z morských slimákov druhu Conus marmoreus a možnosti využitia týchto peptidov v liečbe neuroblastómu pomocou cielenia norepinefrínového transportéru. Päť peptidov založených na tomto konotoxíne bolo simulovaných pomocou simulácii molekulárnej dynamiky, ich trajektórie boli analyzované pre zistenie vlastností týchto peptidov. Dva homologické modely ľudského norepinefrínového transportéru boli vytvorené pre analýzu väzobných vlastností peptidov založených na konotoxíne ku norepinefrínovému transportéru. Peptidy boli následne syntetizované a použité na pokrytie apoferitínových nanočastíc s elipticínom uväzneným vnútri apoferitínu. Vytvorené peptidy a nanočastice boli ďalej skúmané pre objasnenie ich fyzikálo-chemických vlastností. Interakcie a cytotoxicita boli skúmané aplokáciou nanočastíc na bunky neuroblastómu a epitelu. Z in silico a in vitro analýz vyšiel YKL-6 peptid ako najlepší kandidát na ďalší výskum.

Hintergrund. Sibutramin ist ein Medikament, das häufig zur Gewichtsreduktion eingesetzt wird. Es hemmt die Wiederaufnahme von Noradrenalin und Serotonin. Noradrenalin-Wiederaufnahme-Hemmung könnte zu arterieller Hypertonie führen. Methoden. In einer doppelt-blinden, randomisierten Cross-Over Studie untersuchten wir 11 junge, gesunde Probanden (7 Männer, 4 Frauen, Alter 27±2 Jahre, BMI 23.1±0.7Kg/m2). Sie nahmen 26 Stunden (h) und 14h vor Studienbeginn 10mg Sibutramin ein, und 2h vor Untersuchungsbeginn 20mg. Die Wirkung verglichen wir mit Placebo und der Kombination von Sibutramin und 200mg des Beta1-Adrenorezeptor-Blockers Metoprolol. Autonome Reflextests, eine graduelle Kipptischuntersuchung, Plasmakatecholamin-Bestimmungen und eine indirekt kalorimetrische Messung wurden durchgeführt. Herzfrequenz- und Blutdruckbestimmungen erfolgten kontinuierlich und nichtinvasiv. Wir zeichneten das Schlagvolumen, das Herzzeitvolumen und den peripheren Widerstand impedanzkardiografisch auf. Ergebnisse. Sibutramin verursachte einen leichten Anstieg der Herzfrequenz in Ruhe (p / Background. Sibutramine, a serotonin and norepinephrine transporter blocker, is widely used as an adjunctive obesitytreatment. Norepinephrine reuptake inhibition with sibutramine conceivably could exacerbate arterial hypertension andpromote cardiovascular disease. Methods. In 11 healthy subjects (7 men, age 27±2 years, body mass index 23.1±0.7 kg/m2), we compared the effect of sibutramine or matching placebo (ingested 26, 14, and 2 hours before testing) on cardiovascular responses to autonomic reflex tests and to a graded head-up tilt test. In addition, we tested sibutramine in combination with metoprolol. Testing was conducted in a double-blind and crossover fashion. Results. Supine systolic blood pressure was 113±3 mm Hg with placebo, 121±3 mm Hg with sibutramine (P

Die grundlegenden neurobiologischen Mechanismen für das Zusammenwirken eines pathologisch erhöhten Körpergewichts und der gewichts-assoziierten emotionalen Belastung sind bisher noch wenig erforscht. Die vorliegende Arbeit untersucht gezielt Abweichungen der regionalen Transporter-Verfügbarkeiten mittels Positronen-Emissions-Tomographie (PET) mit hochselektiven Marker für den Noradrenalin- (NET) sowie den Serotonin-(5-Hydroxytryptamin-)transporter (5-HTT) und funktioneller Magnetresonanztomographie (fMRT) sowie damit verbundene Alterationen der neuronalen Ruhe-(resting-state-)Aktivität konnektierter Hirnregionen. Die Ergebnisse der kombinierten PET/fMRT Analyse wurden mit zwei neuropsychologischen Scores zur Erfassung allgemeiner und gewichtsabhängiger emotionaler Veränderungen korreliert. Insgesamt 48 Teilnehmer (Body-Mass-Index [BMI]: 19 - 50 kg/m2) erhielten eine fMRT und eine PET mittels NET-selektivem [11C]MRB (n = 20) beziehungsweise 5-HTT-selektivem [11C]DASB (n = 28). Die PET ergab im Hypothalamus eine tendentielle, BMI-abhängig verminderte Verfügbarkeit des NET, nicht jedoch des 5-HTT. Zusätzlich fand sich bei steigendem BMI innerhalb beider Neurotransmitternetzwerke in Abhängigkeit zur jeweiligen Transporter-Verfügbarkeit eine teils geschlechtsspezifisch verstärkte funktionelle Konnektivität zwischen dem Hypothalamus und Hirnregionen der Verarbeitung und Bewertung von Nahrungsreizen. Korrelationen der lokalen resting-state Aktivitäten mit den neuropsychologischen Scores lassen vermuten, dass diese Regionen zudem auch mit langfristigen, negativen Veränderungen des gewichtsabhängigen emotionalen Wohlbefindens assoziiert sind. Insgesamt spielen diese beiden zentralen Neurotransmitter-Systeme eine wichtige Rolle in der Modulation von Netzwerken zur Regulation des gewichtsabhängigen emotionalen Wohlbefindens und könnten somit wichtige Anhaltspunkte für neue pharmakologische Ansätze bereitstellen.

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.