Macrophage-derived netrin-1 drives adrenergic nerve-associated lung fibrosis.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1-expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1's receptor, deleted in colorectal carcinoma; and therapeutic α1 adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1+ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α1 adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1-driven adrenergic processes and introduced α1 blockers as a potentially new fibrotic therapy.

Norepinephrine metabolite DOPEGAL activates AEP and pathological Tau aggregation in locus coeruleus.

Aberrant Tau inclusions in the locus coeruleus (LC) are the earliest detectable Alzheimer's disease-like (AD-like) neuropathology in the human brain. However, why LC neurons are selectively vulnerable to developing early Tau pathology and degenerating later in disease and whether the LC might seed the stereotypical spread of Tau pathology to the rest of the brain remain unclear. Here, we show that 3,4-dihydroxyphenylglycolaldehyde, which is produced exclusively in noradrenergic neurons by monoamine oxidase A metabolism of norepinephrine, activated asparagine endopeptidase that cleaved Tau at residue N368 into aggregation- and propagation-prone forms, thus leading to LC degeneration and the spread of Tau pathology. Activation of asparagine endopeptidase-cleaved Tau aggregation in vitro and in intact cells was triggered by 3,4-dihydroxyphenylglycolaldehyde, resulting in LC neurotoxicity and propagation of pathology to the forebrain. Thus, our findings reveal that norepinephrine metabolism and Tau cleavage represent the specific molecular mechanism underlying the selective vulnerability of LC neurons in AD.

Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow.

Clinical observations have identified an association between psychologic stress and cancer relapse, suggesting that the sympathetic nervous system/norepinephrine (NE) plays a role in reactivation of dormant disseminated tumor cells (DTC) in the bone marrow niche. Here, the mechanism by which NE regulates prostate cancer DTCs in the marrow is explored. NE directly stimulated prostate cancer cell proliferation through ß2-adrenergic receptors (ADRB2). NE also altered prostate cancer proliferation in the marrow niche by indirectly downregulating the secretion of the dormancy inducing molecule growth arrest specific-6 (GAS6) expressed by osteoblasts. These observations were confirmed in cocultures of prostate cancer cells expressing the fluorescent ubiquitination-based cell-cycle reporters (FUCCI) and osteoblasts isolated from GAS6-deficient (GAS6-/-) animals. A novel ex vivo model system, using femurs harvested from GAS6+/+ or GAS6-/- mice, was used to confirm these results. As in coculture, when prostate cancer cells were injected into the marrow cavities of GAS6+/+ femurs, NE altered the prostate cancer cell cycle. However, NE had less of an impact on prostate cancer cells in femur explants isolated from GAS6-/- mice. Together, this study demonstrates that NE reactivates prostate cancer cell cycling through both a direct action on prostate cancer cells and indirectly on adjacent niche cells.Implications: Identification of mechanisms that target DTCs may provide novel therapeutic approaches to prevent or treat cancer metastases more effectively. Mol Cancer Res; 15(12); 1644-55. ©2017 AACR.

Sympathetic denervation-induced MSC mobilization in distraction osteogenesis associates with inhibition of MSC migration and osteogenesis by norepinephrine/adrb3.

The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE) in distraction-induced bone callus, down-regulate ß3-adrenergic receptor (adrb3) in bone marrow mesenchymal stem cells (MSCs), and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1)-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2) and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3). Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor-2 (RUNX2), but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes.

Low dopamine function in attention deficit/hyperactivity disorder: should genotyping signify early diagnosis in children?

Attention deficit/hyperactivity disorder (ADHD) is present in 8% to 12% of children, and 4% of adults worldwide. Children with ADHD can have learning impairments, poor selfesteem, social dysfunction, and an increased risk of substance abuse, including cigarette smoking. Overall, the rate of treatment with medication for patients with ADHD has been increasing since 2008, with ≥ 2 million children now being treated with stimulants. The rise of adolescent prescription ADHD medication abuse has occurred along with a concomitant increase of stimulant medication availability. Of adults presenting with a substance use disorder (SUD), 20% to 30% have concurrent ADHD, and 20% to 40% of adults with ADHD have a history of SUD. Following a brief review of the etiology of ADHD, its diagnosis and treatment, we focus on the benefits of early and appropriate testing for a predisposition to ADHD. We suggest that by genotyping patients for a number of known, associated dopaminergic polymorphisms, especially at an early age, misdiagnoses and/or over-diagnosis can be reduced. Ethical and legal issues of early genotyping are considered. As many as 30% of individuals with ADHD are estimated to either have secondary side-effects or are not responsive to stimulant medication. We also consider the benefits of non-stimulant medication and alternative treatment modalities, which include diet, herbal medications, iron supplementation, and neurofeedback. With the goals of improving treatment of patients with ADHD and SUD prevention, we encourage further work in both genetic diagnosis and novel treatment approaches.

Dopaminergic and noradrenergic gene polymorphisms and response to methylphenidate in korean children with attention-deficit/hyperactivity disorder: is there an interaction?

OBJECTIVE: We aimed to investigate the independent and interaction effects of dopamine transporter gene (DAT1), dopamine D4 receptor gene (DRD4), alpha-2A adrenergic receptor gene (ADRA2A), and norepinephrine transporter gene (NET1), with regard to treatment response to methylphenidate (MPH) in attention-deficit/hyperactivity disorder (ADHD). METHODS: The participants of the study were 103 children and adolescents (ages 9.1±2.1 years) diagnosed as having ADHD according to American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV) criteria. They were enrolled in an 8-week, open-label trial of MPH. The good responder group was defined as subjects having an ≥50% decrease in the ADHD Rating Scale-IV (ADHD-RS) total score from the baseline, and at the same time a Clinical Global Impressions-Improvement Scale (CGI-I) score of 1 or 2, both at the 8th week of MPH treatment. Multivariate stepwise logistic regression was performed to examine the independent and interaction effects of genotypes on the dichotomized MPH treatment response. RESULTS: Significant interaction effects on MPH response were detected between the genotypes of the DRD4 variable number of tandem repeat (VNTR) polymorphisms and those of either the ADRA2A DraI or the NET1 -3081(A/T) polymorphisms; significant interaction effects were also detected between the genotypes of the ADRA2A DraI polymorphisms and those of either the NET1 G1287A or the NET1 -3081(A/T) polymorphisms (Nagelkerke R(2)=0.40). No significant independent effect of a genotype was detected according to the stepwise logistic regression results. CONCLUSION: The results suggest that genes involved in the dopaminergic and noradrenergic systems might interact to form important predictors of short-term response to MPH.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes.

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

Pancreatic cancer cells and normal pancreatic duct epithelial cells express an autocrine catecholamine loop that is activated by nicotinic acetylcholine receptors α3, α5, and α7.

Pancreatic cancer is the fourth leading cause of cancer deaths in developed countries. Smoking is an established risk factor for this malignancy but the underlying mechanisms are poorly understood. Previous reports have provided evidence that nicotinic acetylcholine receptors (nAChR) and beta adrenergic receptors (ß-AR) stimulate the growth and migration of pancreatic cancer cells. However, a potential cooperation of these two receptor families in the regulation of pancreatic cancer has not been studied to date. Using two pancreatic cancer cell lines and immortalized pancreatic duct epithelia in vitro, our current data show that all three cell lines synthesized and released the catecholamine neurotransmitters noradrenaline and adrenaline upon exposure to nicotine and that this activity was regulated by α3, α5, and α7-nAChRs. In accordance with the established function of these catecholamines as ß-AR agonists, nicotine-induced cell proliferation was blocked by the ß-AR antagonist propranolol. Nicotine-induced proliferation was also abolished by the α7-nAChR antagonist α-bungarotoxin, whereas catecholamine production in response to nicotine was blocked by gene knockdown of the α3, α5, and α7-nAChRs. The nicotinic agonists acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK induced the phosphorylation of CREB, ERK, Src, and AKT and these responses were inhibited by propranolol. Our findings identify this hitherto unknown autocrine catecholamine loop as an important regulatory cascade in pancreatic cancer that may prove a promising new target for cancer intervention.

ß-1 and ß-2 adrenergic receptor polymorphism and association with cardiovascular response to orthostatic screening.

Variation in the beta-1 and beta-2 adrenergic receptor genes (ADRB1 and ADRB2, respectively) may influence cardiovascular reactivity including orthostatic stress. We tested this hypothesis in a head-up tilt (HUT) screening protocol in healthy young adults without history of syncope. Following brachial arterial catheter insertion, 120 subjects (age 18-40, 72 females, Caucasian) underwent 5min 60° HUT. Polymorphisms tested were: Ser49/Gly and Arg389/Gly in ADRB1; and Arg16/Gly, Gln27/Glu, and Thr164/Ile in ADRB2. Three statistical models (recessive, dominant, additive) were evaluated using general linear models with analysis for each physiologic variable. A recessive model demonstrated a significant association between Arg16/Gly and: absolute supine and upright HR; HUT-induced change in cardiac index (CI), stroke index (SI) and systemic vascular resistance (SVR); and supine and upright norepinephrine values. Blood pressure was not influenced by genotype. Fewer associations were present for other polymorphisms: Ser49/Gly and the change in SI (dominant model), and Arg389/Gly and supine and HUT norepinephrine (additive model). We conclude that in this population, there is a robust association between Arg16/Gly and HUT responses, such that 2 copies of Arg16 increase supine and upright HR, and greater HUT-induced decreases in CI and SI, with greater increases in SVR and norepinephrine. ADRB1 gene variation appears to impact SI and plasma NE levels but not HR. Whether ADRB2 gene variation is ultimately disease-causing or disease-modifying, this study suggests an association between Arg16/Gly and postural hemodynamics, with sympathetic noradrenergic activity affected in a similar direction. This may have implications in the development of orthostatic disorders.

Norepinephrine and ß1-adrenergic signaling facilitate activation of hippocampal CA1 pyramidal neurons during contextual memory retrieval.

We previously described a role for adrenergic signaling in the hippocampus to promote contextual and spatial memory retrieval. A subsequent study performing expression analysis of the immediate-early gene (IEG) Arc suggested that activation of CA1 but not CA3 pyramidal neurons during memory retrieval is impaired in the absence of NE. The current study sought to confirm and extend those observations by performing expression analysis of a second IEG product, Fos, following a much greater variety of testing conditions. In mutant mice lacking NE, induction of Fos was normal in all regions of the hippocampus and amygdala shortly after fear conditioning. In contrast, when testing contextual fear 1 day after training, induction of Fos in CA1 and the central nucleus of the amygdala (CeA), but not CA3, the dentate gyrus or other amygdaloid nuclei, was impaired in the mutant mice. This pattern corresponded to the memory retrieval deficit exhibited by these mice. On the other hand, induction was normal in CA1 and CeA when testing cued fear 1 day after training, or contextual fear 1 week or 1 month after training, conditions in which retrieval are normal in the absence of NE. Acute restoration of NE in the mutant mice before testing but not before training rescued retrieval of contextual fear and restored Fos induction in CA1 and CeA. Because NE facilitates retrieval through the activation of ß(1)-adrenergic receptors, ß(1) knockout mice were also examined and found to exhibit reduced induction of Fos in CA1 and CeA following retrieval. Based on these and previous results, we hypothesize that adrenergic signaling is critical for the full activation of CA1 pyramidal neurons in response to excitatory input from CA3 pyramidal neurons conveying retrieved contextual information.

Thy-1 mRNA destabilization by norepinephrine a 3' UTR cAMP responsive decay element and involves RNA binding proteins.

Thy-1 is a cell surface protein important in immunologic and neurologic processes, including T cell activation and proliferation, and neuronal outgrowth. In murine thymocytes, Thy-1 is downregulated in response to norepinephrine (NE) through posttranscriptional destabilization of its mRNA mediated by ßAR/AC/cAMP/PKA signaling. In this study we investigated factors involved in NE/cAMP-mediated Thy-1 mRNA destabilization in S49 thymoma cells, and identified a region containing two copies of the AUUUA regulatory element (ARE), a motif commonly associated with mRNA decay, in the Thy-1 mRNA 3' UTR. Insertion of the Thy-1 ARE region into a reporter gene, resulted in cAMP induced destabilization of the reporter gene mRNA. RNA-protein binding studies revealed multiple Thy-1 ARE binding proteins, including AUF1, HuR, and TIAR. RNA silencing of HuR enhanced cAMP-mediated downregulation of Thy-1 mRNA, in contrast, silencing AUF1 had no effect. Immunoblotting revealed multiple proteins phosphorylated by PKA as a result of NE or cAMP signaling. These results reveal that the machinery of NE/cAMP modulation of Thy-1 mRNA decay involves a cAMP responsive ARE in its 3' UTR and multiple site specific ARE binding proteins. These findings add to our knowledge of Thy-1 mRNA regulation and provide insight into the regulation of ARE containing mRNAs, which impacts stress-related immunosuppression.

Relationship between concentrations of progesterone, oxytocin, noradrenaline, gene expression and protein level for their receptors in corpus luteum during estrous cycle in the cow.

The aim of these studies was to evaluate of the relationship between luteal concentrations of oxytocin (OT), noradrenaline (NA), progesterone (P4), oxytocin receptors (OT-R) and beta(2)-adrenoreceptors (beta(2)-R) gene expression and their protein level throughout the estrous cycle in cattle. Corpora lutea (CL) collected during days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle were used in these studies. Concentrations of P4, OT and NA were determined in tissue extracts. Gene expression and protein level for OT-R and beta(2)-R were investigated by RT-PCR and Western blot, respectively. Luteal concentration of P4 was higher (P<0.01) on days 6-10 and 11-16 than during days 1-5 and 17-20 of the estrous cycle. Concentration of OT was the highest on days 1-5 and 6-10 of the estrous cycle. This concentration decreased (P<0.01) during days 11-16 reaching the lowest level (P<0.001) on days 17-20 as opposed to days 1-10 of the estrous cycle. Expression of OT-R mRNA was lower on days 6-16 (P<0.05) followed by its increase on days 17-20 as opposed to the expression observed on days 1-5. Expression of OT-R mRNA was negatively correlated (P<0.05) with the profile of OT-R protein level. This latter parameter was the lowest on days 17-20 and 1-5, and the highest on days 6-10 and 11-16. Oxytocin concentration was negatively correlated (P<0.05) with expression of OT-R mRNA and positively correlated (P<0.001) with OT-R protein level. This protein level was only correlated with P4 (r=0.59; P<0.05). Concentration of OT was positively correlated with level of P4 (P<0.001). Concentration of NA was the highest on days 1-5 of the estrous cycle, whereas it was similar or lower (P<0.05) on days 6-21. Expression of mRNA for beta(2)-R was the lowest on days 1-5 and was highly increased (P<0.05) on days 6-16. The expression was the highest (P<0.001) on days 17-21 compared to all others group of the estrous cycle. Protein level for beta(2)-R was the highest on days 1-5 and decreased (P<0.05) on days 6-10 and 11-16. The expression was the lowest on days 17-20 compared to the beta(2)-R protein level in CL from all others stages of the estrous cycle. Protein level for beta(2)-R was positively correlated (P<0.05) with the OT concentration. Expression of mRNA for beta(2)-R was negatively correlated (P<0.001) with level of beta(2)-R protein. No correlation was found between beta(2)-R mRNA expression and NA concentration or between NA and P4 concentrations. Results presented in this study suggest an evident relationship between OT and NA and may play an important role in the regulation of luteal steroidogenesis during all stages of the estrous cycle.

Biochemistry of postmortem brains in Parkinson's disease: historical overview and future prospects.

Biochemical studies on postmortem brains of patients with Parkinson's disease (PD) have greatly contributed to our understanding of the molecular pathogenesis of this disease. The discovery by 1960 of a dopamine deficiency in the nigro-striatal dopamine region of the PD brain was a landmark in research on PD. At that time we collaborated with Hirotaro Narabayashi and his colleagues in Japan and with Peter Riederer in Germany on the biochemistry of PD by using postmortem brain samples in their brain banks. We found that the activity, mRNA level, and protein content of tyrosine hydroxylase (TH), as well as the levels of the tetrahydrobiopterin (BH4) cofactor of TH and the activity of the BH4-synthesizing enzyme, GTP cyclohydrolase I (GCHI), were markedly decreased in the substantia nigra and striatum in the PD brain. In contrast, the molecular activity (enzyme activity/enzyme protein) of TH was increased, suggesting a compensatory increase in the enzyme activity. The mRNA levels of all four isoforms of human TH (hTH1-hTH4), produced by alternative mRNA splicing, were also markedly decreased. This finding is in contrast to a completely parallel decrease in the activity and protein content of dopamine beta-hydroxylase (DBH) without changes in its molecular activity in cerebrospinal fluid (CSF) in PD. We also found that the activities and/or the levels of the mRNA and protein of aromatic L-amino acid decarboxylase (AADC, DOPA decarboxylase), DBH, phenylethanolamine N-methyltransferase (PNMT), which synthesize dopamine, noradrenaline, and adrenaline, respectively, were also decreased in PD brains, indicating that all catecholamine systems were widely impaired in PD brains. Programmed cell death of the nigro-striatal dopamine neurons in PD has been suggested from the following findings on postmortem brains: (1) increased levels of pro-inflammatory cytokines such as TNF-alpha and IL-6; (2) increased levels of apoptosis-related factors such as TNF-alpha receptor R1 (p 55), soluble Fas and bcl-2, and increased activities of caspases 1 and 3; and (3) decreased levels of neurotrophins such as brain-derived nerve growth factor (BDNF). Immunohistochemical data and the mRNA levels of the above molecules in PD brains supported these biochemical data. We confirmed by double immunofluorescence staining the production of TNF-alpha and IL-6 in activated microglia in the putamen of PD patients. Owing to the recent development of highly sensitive and wide-range analytical methods for quantifying mRNAs and proteins, future assays of the levels of various mRNAs and proteins not only in micro-dissected brain tissues containing neurons and glial cells, but also in single cells from frozen brain slices isolated by laser capture micro-dissection, coupled with toluidine blue, Nissl staining or immunohistochemical staining, should further contribute to the elucidation of the molecular pathogenesis of PD and other neurodegenerative or neuropsychiatric diseases.

[Meta-analysis of candidate genes in attention-deficit hyperactivity disorder]. / Meta-analyse des gènes candidats dans le trouble déficit attentionnel avec hyperactivité (TDAH).

Attention-deficit hyperactivity disorder (ADHD) is a common behavioral disorder observed during childhood, detected in 3% to 5% of school-age children. The disorder is characterised by marked inattention, hyperactivity, and impulsiveness. In most cases, symptoms can be treated by catecholamine-releasing drugs, such as methylphenidate. Children with ADHD are at higher risk for substance abuse and oppositional, conduct and mood disorders. Familial and adoption studies shed light on the genetic vulnerability of ADHD. Twin studies estimated the broad heritability to range between 40% and 90%. The mode of transmission is yet unknown, but is likely polygenic. Molecular genetic studies in ADHD should contribute to a greater understanding of the pathophysiology of the disorder (genetics of the vulnerability), and could help to select a more rational type of treatment (pharmacogenetic). Family-based association studies already performed are reviewed in this manuscript. Association studies, using haplotype relative risk (HRR) or transmission disequilibrium test (TDT) have focused on candidate genes which code for proteins potentially involved in the etiopathogenesis of the disorder. Genes involved in dopamine, serotonin, and noradrenalin systems have thus been assessed for their role in core features of ADHD, such as motor overactivity, inattention, and impulsiveness. According to a meta-analysis, the DAT1 gene, an obvious candidate gene in ADHD vulnerability, does not appear to be involved (OR = 1.13, p = 0.21). On the other hand, DRD4 (OR = 1.26, p = 0.01) and DRD5 (OR = 1.4, p = 0.01) are significantly associated to ADHD according to the present meta-analysis, confirming previous ones. Recent studies showed a trend for an association between one allele of the 5-HTT (considering case-control studies) and DBH (OR = 1.27, p = 0.06) genes and ADHD, but these positive findings have to be replicated. ADHD is a complex disorder with potentially many different risk factors. Genetic and phenotypic heterogeneity could explain why some association studies are positive, whereas others are negative. For instance, different developmental pathways are likely to lead to similar clinical outcomes. More clear-cut phenotypes, such as ADHD with conduct disorder, or ADHD with bipolar disorder, could be more homogenous, the genes involved being therefore more easy to detect. These phenotypes are beginning to be specifically studied in molecular genetics. In addition, the development of pharmacogenetics could help to identify predictors of clinical response for a specific type of treatment, which would be clearly helpful in clinical practice.

Influence of monoamines on differentiating gonadotropin-releasing hormone neurones in foetal mice.

This study evaluated the influence of monoamines, serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline, on differentiating gonadotropin-releasing hormone (GnRH)-producing neurones in foetal mice. The differentiation and migration of GnRH neurones were compared in Tg8 mice (the knocked-out gene encoding monoamine oxidase A) with increased levels of 5-HT and noradrenaline and in C3H mice with normal metabolism of monoamines in C3H mice. To achieve this, immunocytochemistry for GnRH combined with quantitative and semiquantitative image analysis were employed. GnRH neurones in foetuses at the 18th embryonic day were detected in the forebrain along the trajectory of their migration from the olfactory bulbs to the hypothalamic retrochiasmatic region. The total number of GnRH neurones in the forebrain in knockout mice was significantly lower compared to C3H mice, suggesting an inhibiting influence of monoamines on the proliferation of precursor cells. The fraction of GnRH neurones in the caudal part of the trajectory of their migration in Tg8 mice exceeded significantly those in C3H foetuses, whereas there was a reverse in the rostral part of the trajectory. These data suggest that an excess of 5-HT and noradrenaline served to accelerate the GnRH neurone migration in Tg8 mice. Moreover, an excess of 5-HT and noradrenaline provided a minor effect on the area and optical density of GnRH neurones (i.e. on GnRH neurone differentiation). Thus, an excess of 5-HT and noradrenaline appears to inhibit the proliferation of the precursor cells of GnRH neurones and stimulates the GnRH neurone migration to the place of their final location in the septo-preoptic region.

Aldosterone receptor blockade improves left ventricular remodeling and increases ventricular fibrillation threshold in experimental heart failure.

OBJECTIVES: To investigate the effects of aldosterone receptor blockade in postinfarction heart failure. METHODS: Eighty-seven rats with moderate myocardial infarction were randomized to receive either no drug or canrenone, the active metabolite of spironolactone, 20 mg/kg/day, or ramipril, 1 mg/kg/day, or a combination of the two drugs. Treatment was initiated 1 month after coronary ligation and lasted 4 weeks. Echocardiography was performed at baseline and after 4 weeks. LV catheterization, isolated heart studies, morphometric histology, myocardial norepinephrine and SERCA-2 mRNA were assessed at the end of the treatment period. RESULTS: Infarct sizes were 33+/-3, 32+/-3, 34+/-3, and 34+/-4% in the placebo, canrenone, ramipril, and combination groups, respectively. Canrenone attenuated LV remodeling, improved LV systolic and diastolic function, and markedly reduced interstitial and perivascular fibrosis. These effects were increased by concomitant ramipril therapy. Moreover, myocardial norepinephrine content was decreased while ventricular fibrillation threshold significantly augmented by canrenone. SERCA-2 levels remained unchanged. CONCLUSIONS: Canrenone attenuated LV dilation and interstitial remodeling, and improved LV filling dynamics and systolic function in the rat model of postinfarction heart failure. Addition of ramipril conferred further cardioprotection. Canrenone also reduced myocardial norepinephrine content and increased ventricular fibrillation threshold. The data provide a potential explanation for the decreased sudden death observed in the RALES study. The mechanisms of action of aldosterone inhibition are still poorly understood, despite its proven efficacy in heart failure. Rats with postinfarction heart failure were randomized to receive for 1 month either no drug or canrenone, or ramipril, or a combination of canrenone and ramipril. Canrenone treatment was associated with a significant attenuation of LV dilation, better LV diastolic and systolic dynamics, and a marked reduction of reactive fibrosis. These effects were enhanced by concomitant ramipril therapy. Moreover, canrenone increased ventricular fibrillation threshold and reduced myocardial norepinephrine content. The data may explain the reduced mortality demonstrated by the RALES.

Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways.

Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland. In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses. We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons. CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones. CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture. CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases. The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays. We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways.

6-fluorodopamine selectively destroys neuroblastoma cells expressing the noradrenaline transporter.

BACKGROUND: 6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells. PROCEDURE: We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells. RESULTS: Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA. CONCLUSIONS: We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells.

A gene therapy approach to enhance the targeted radiotherapy of neuroblastoma.

BACKGROUND: The aims of this study were to determine whether the introduction and expression of the noradrenaline transporter (NAT) gene into NAT-negative neuroblastoma cell lines would make them amenable to targeted radiotherapy using [(131)I]MIBG. PROCEDURE: Neuroblastoma cell lines were transfected with a eukaryotic expression vector containing the bovine noradrenaline transporter cDNA under the expression of the CMV promoter. Stable transfectants were created by selection in geneticin (G418) and were characterised for their MIBG uptake ability and susceptibility to [(131)I]MIBG therapy. RESULTS: The cell line SK-N-MC, which normally shows no ability to take up MIBG, was successfully transfected with bNAT. SK-N-MC.bNAT transfectants exhibited uptake and release kinetics similar to those of the natural NAT-expressing cell line SK-N-BE(2c). Levels of [(131)I]MIBG uptake were 33% of those of the highest naturally NAT-expressing cell line SK-N-BE(2c). Growth delay assays using multicellular spheroids indicated that this degree of [(131)I]MIBG uptake was sufficient to inhibit growth at radioactive concentrations of 4 Mbq/ml. CONCLUSIONS: These results demonstrate the feasibility of combining gene therapy with targeted radiotherapy to enhance uptake, and hence radiation dose, to neuroblastoma tumours using [(131)I]MIBG. With the appropriate delivery vehicle and tumour-specific control of expression, the introduction of noradrenaline transporter molecules may be a viable means of enhancing the response of neuroblastoma tumours to [(131)I]MIBG therapy.

Norepinephrine-deficient mice have increased susceptibility to seizure-inducing stimuli.

Several lines of evidence suggest that norepinephrine (NE) can modulate seizure activity. However, the experimental methods used in the past cannot exclude the possible role of other neurotransmitters coreleased with NE from noradrenergic terminals. We have assessed the seizure susceptibility of genetically engineered mice that lack NE. Seizure susceptibility was determined in the dopamine beta-hydroxylase null mutant (Dbh -/-) mouse using four different convulsant stimuli: 2,2,2-trifluroethyl ether (flurothyl), pentylenetetrazol (PTZ), kainic acid, and high-decibel sound. Dbh -/- mice demonstrated enhanced susceptibility (i.e., lower threshold) compared with littermate heterozygous (Dbh +/-) controls to flurothyl, PTZ, kainic acid, and audiogenic seizures and enhanced sensitivity (i.e., seizure severity and mortality) to flurothyl, PTZ, and kainic acid. c-Fos mRNA expression in the cortex, hippocampus (CA1 and CA3), and amygdala was increased in Dbh -/- mice in association with flurothyl-induced seizures. Enhanced seizure susceptibility to flurothyl and increased seizure-induced c-fos mRNA expression were reversed by pretreatment with L-threo-3, 4-dihydroxyphenylserine, which partially restores the NE content in Dbh -/- mice. These genetically engineered mice confirm unambiguously the potent effects of the noradrenergic system in modulating epileptogenicity and illustrate the unique opportunity offered by Dbh -/- mice for elucidating the pathways through which NE can regulate seizure activity.