Noradrenergic Nerve Terminals Research Articles

Zamicastat is a noncompetitive Dopamine‐β‐hydroxylase inhibitor that modulates sympathetic nervous system activity

Dopamine‐β‐hydroxylase (DβH; EC 1.14.17.1) is an enzyme responsible for the conversion of dopamine (DA) into norepinephrine (NE). It is present in the chromaffin cells of adrenal medulla and in noradrenergic nerve terminals of the central and peripheral sympathetic nervous system. DβH inhibition has been proposed as an alternative mechanism for the treatment of hypertension and chronic heart failure. Zamicastat ((R)‐5‐(2‐(benzylamino)ethyl)‐1‐(6,8‐difluorochroman‐3‐yl)‐1H‐imidazole‐2(3H)‐thione) is a novel DβH inhibitor. The aim of this study was to characterize the pharmacodynamics of Zamicastat in vivo, both in Wistar Han and in spontaneously hypertensive rats (SHR), and on DβH in human cells. DβH activity was measured by a modification of the Nagatsu and Udenfriend (1) spectrophotometric method, based on the conversion of tyramine into octopamine either in adrenal tissues of rats administered orally with Zamicastat (30 mg/Kg) or vehicle, or in human neuroblastoma (SK‐N‐SH) cells. Zamicastat pharmacodynamic characterization in vivo was performed in Wistar rats by evaluating NE and DA levels in tissues using HPLC with electrochemical detection at 9h post‐administration. Additionally, systolic and diastolic blood pressure, heart rate and activity were measured continually by telemetry in SHR rats (a model of essential hypertension) and their controls, Wistar Kyoto (WKY). Zamicastat was found to be a reversible noncompetitive inhibitor of DβH towards tyramine, with a Ki value of 43 (21; 66) nM and either noncompetitive or uncompetitive towards ascorbic acid. Zamicastat (30 mg/Kg, p.o.) fully inhibits adrenal DβH activity from 2 to 8h recovering to baseline values after 24h. This is accompanied with a decrease in NE and an increase in DA in peripheral tissues, namely left ventricle and kidney but not significantly in the central nervous system (brain stem or prefrontal‐cortex). Zamicastat at 3, 30 and 100 mg/Kg displayed a dose‐dependent decrease on systolic and diastolic blood pressure with no effect in heart rate or overall activity when evaluated in SHR rats by telemetry. In conclusion, Zamicastat is a DβH inhibitor displaying binding affinity for both the free enzyme and the complex enzyme‐substrate, that when orally administered at 30 mg/Kg to rats acts peripherally and significantly reduces sympathetic activity by decreasing NE levels and consequently systolic and diastolic blood pressure in an animal model of essential hypertension.

Isolation and Molecular Identification of a Novel Tyramine-producing Bacterium, <i>Rummeliibacillus pycnus</i>

Decarboxylation of tyrosine through tyrosine decarboxylase enzyme produces tyramine which may represent a serious threat to public health, as it may cause severe toxicological effects. Intake of tyramine will invariably result in acute symptoms as it can rapidly gain access to the bloodstream and to various organs where it can act as a vasoactive agent through interaction with the sympathetic noradrenergic nerve terminals innervating cardiac and vascular smooth muscle tissues. This research is focusing on tyramine as one of the indicators of inappropriate food storage and processing conditions of milk products. In the current study, 25 dairy product samples were collected from local markets in Alexandria, Egypt, and were analyzed for bacterial contamination and prevalence of tyramine-producing bacteria by PCR using degenerate primers (DEC5/DEC3). While cream was the most contaminated dairy product, balady yogurt samples were the least contaminated ones. Different types of tyraminogenic bacteria have been isolated from the collected dairy products. Cream samples were contaminated with the widest variety of tyraminogenic bacteria among the isolated samples including Bacillus pumilis, Escherichia coli, Enterococcus faecium, and Proteus mirabilis. A total of 35 strains harboring tyrosine decarboxylase gene were detected with the identification of a novel tyramine-producing strain: Rummeliibacillus pycnus. These results indicate the promising application of degenerate primers (DEC5/DEC3) to detect tyramine production in dairy products, a goal that has been regarded as a challenge by manufacturers.

Dynamic monitoring of single-terminal norepinephrine transporter rate in the rodent cardiovascular system: A novel fluorescence imaging method.

Here, we validate the use of a novel fluorescent norepinephrine transporter (NET) substrate for dynamic measurements of transporter function in rodent cardiovascular tissue; this technique avoids the use of radiotracers and provides single-terminal resolution.Rodent (Wistar rats and C57BL/6 mice) hearts and mesenteric arteries (MA) were isolated, loaded with NET substrate Neurotransmitter Transporter Uptake Assay (NTUA) ex vivo and imaged with confocal microscopy.NTUA labelled noradrenergic nerve terminals in all four chambers of the heart and on the surface of MA. In all tissues, a temperature-dependent, stable linear increase in intra-terminal fluorescence upon NTUA exposure was observed; this was abolished by NET inhibitor desipramine (1 μM) and reversed by indirectly-acting sympathomimetic amine tyramine (10 μM). NET reuptake rates were similar across the mouse cardiac chambers. In both species, cardiac NET activity was significantly greater than in MA (by 62 ± 29% (mouse) and 21 ± 16% (rat)). We also show that mouse NET reuptake rate was twice as fast as that in the rat (for example, in the heart, by 94 ± 30%). Finally, NET reuptake rate in the mouse heart was attenuated with muscarinic agonist carbachol (10 μM) thus demonstrating the potential for parasympathetic regulation of norepinephrine clearance.Our data provide the first demonstration of monitoring intra-terminal NET function in rodent cardiovascular tissue. This straightforward method allows dynamic measurements of transporter rate in response to varying physiological conditions and drug treatments; this offers the potential to study new mechanisms of sympathetic dysfunction associated with cardiovascular disease.

In-vivo staging of pathology in REM sleep behaviour disorder: a multimodality imaging case-control study

Accumulating evidence suggests that α-synuclein aggregates-a defining pathology of Parkinson's disease-display cell-to-cell transmission. α-synuclein aggregation is hypothesised to start in autonomic nerve terminals years before the appearance of motor symptoms, and subsequently spread via autonomic nerves to the spinal cord and brainstem. To assess this hypothesis, we investigated sympathetic, parasympathetic, noradrenergic, and dopaminergic innervation in patients with idiopathic rapid eye movement (REM) sleep behaviour disorder, a prodromal phenotype of Parkinson's disease. In this prospective, case-control study, we recruited patients with idiopathic REM sleep behaviour disorder, confirmed by polysomnography, without clinical signs of parkinsonism or dementia, via advertisement and through sleep clinics in Denmark. We used 11C-donepezil PET and CT to assess cholinergic (parasympathetic) gut innervation, 123I-metaiodobenzylguanidine (MIBG) scintigraphy to measure cardiac sympathetic innervation, neuromelanin-sensitive MRI to measure integrity of pigmented neurons of the locus coeruleus, 11C-methylreboxetine (MeNER) PET to assess noradrenergic nerve terminals originating in the locus coeruleus, and 18F-dihydroxyphenylalanine (DOPA) PET to assess nigrostriatal dopamine storage capacity. For each imaging modality, we compared patients with idiopathic REM sleep behaviour disorder with previously published reference data of controls without neurological disorders or cognitive impairment and with symptomatic patients with Parkinson's disease. We assessed imaging data using one-way ANOVA corrected for multiple comparisons. Between June 3, 2016, and Dec 19, 2017, we recruited 22 consecutive patients with idiopathic REM sleep behaviour disorder to the study. Compared with controls, patients with idiopathic REM sleep behaviour disorder had decreased colonic 11C-donepezil uptake (-0·322, 95% CI -0·112 to -0·531; p=0·0020), 123I-MIBG heart:mediastinum ratio (-0·508, -0·353 to -0·664; p<0·0001), neuromelanin-sensitive MRI locus coeruleus:pons ratio (-0·059, -0·019 to -0·099; p=0·0028), and putaminal 18F-DOPA uptake (Ki; -0·0023, -0·0009 to -0·0037; p=0·0013). No between-group differences were detected between idiopathic REM sleep behaviour disorder and Parkinson's disease groups with respect to 11C-donepezil (p=0·39), 123I-MIBG (p>0·99), neuromelanin-sensitive MRI (p=0·96), and 11C-MeNER (p=0·56). By contrast, 15 (71%) of 21 patients with idiopathic REM sleep behaviour disorder had 18F-DOPA Ki values within normal limits, whereas all patients with Parkinson's disease had significantly decreased 18F-DOPA Ki values when compared with patients with idiopathic REM sleep behaviour disorder (p<0·0001). Patients with idiopathic REM sleep behaviour disorder had fully developed pathology in the peripheral autonomic nervous system and the locus coeruleus, equal to that in diagnosed Parkinson's disease. These patients also showed noradrenergic thalamic denervation, but most had normal putaminal dopaminergic storage capacity. This caudorostral gradient of dysfunction supports the hypothesis that α-synuclein pathology in Parkinson's disease initially targets peripheral autonomic nerves and then spreads rostrally to the brainstem. Lundbeck Foundation, Jascha Foundation, and the Swiss National Foundation.

Dopamine‐β‐monooxygenase inhibitors obtained by structure based methods exhibited anti‐hypertensive effect in L‐NAME induced hypertensive rats

IntroductionHypertension is the leading cause of mortality worldwide (Kwan G.F. et. al., Circulation, 2016). While classical anti‐hypertensives like ACE inhibitors, ARBs, calcium channel blockers, beta blockers and diuretics are widely used, they have their own limitations and no new type of drugs emerged in the last few years that use body's own blood pressure lowering mechanism. Dopamine β‐monooxygenase (DBM), expressed in noradrenergic nerve terminals as well as in adrenal medullary chromaffin cells, which convert dopamine into norepinephrine, is a novel target whose inhibition has been shown to help the treatment of hypertension with several advantages over the classical antihypertensives. However, DBM inhibitors are few in number, often result in side‐effects and are frequently non‐responsive to specific population, probably since the known inhibitors so far have been designed on ligand based methods. We hypothesize that rational and three dimensional structure based identification of novel inhibitors may overcome such problems.ObjectiveTo identify novel inhibitors of DBM and evaluate their anti‐hypertensive properties in L‐NAME induced hypertensive rat model.Design and methodAn experimentally validated computational model for human DBM, built in our lab, was used for structure‐based, rational drug design. The three‐dimensional model was used for virtual screening against various small molecule databases. Identified top hits were then tested in vitro against DBM with known inhibitors nepicastat and disulfiram as controls. Binding of the inhibitors to DBM were validated using fluorescence and CD spectroscopy as well as ITC. Pharmacokinetic analysis was performed computationally. Cyto‐ and hemo‐toxicities of the lead compounds were assessed ex vivo. Finally. their antihypertensive efficacies were evaluated in L‐NAME induced hypertensive rat model.ResultsVirtual screening of various compound libraries revealed 69 hits which were then assessed in vitro using a repertoire of biochemical and biophysical methods. UDSC‐171, 180 and 142 were discovered to be potent inhibitors of DBM with IC50s of 1μM, 5.5μM and 18μM, respectively. The inhibitors displayed KD values against DBM in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated that the molecules are unable to cross the BBB. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK293 cell line and insignificant hemo‐toxicities against RBCs. These three leads were successful in preventing elevated systolic blood pressure in L‐NAME induced hypertensive rat models.ConclusionsPresent study successfully developed rapid, systematic and non‐expensive biophysical, biochemical and computational tools for the identification, characterization and validation of DBM inhibitors. Moreover, UDSC‐171, 180 and 142 have been discovered and validated as novel antihypertensives against L‐NAME induced hypertensive rats. (Indian Patent filed for the present study with app. no. 201711036983)Support or Funding InformationDepartment of Biotechnology, Govt. of India

The binding of botulinum neurotoxins to different peripheral neurons

Botulinum neurotoxins are the most potent toxins known. The double receptor binding modality represents one of the most significant properties of botulinum neurotoxins and largely accounts for their incredible potency and lethality. Despite the high affinity and the very specific binding, botulinum neurotoxins are versatile and multi-tasking toxins. Indeed they are able to act both at the somatic and at the autonomic nervous system.In spite of the preference for cholinergic nerve terminals botulinum neurotoxins have been shown to inhibit to some extent also the noradrenergic postganglionic sympathetic nerve terminals and the afferent nerve terminals of the sensory neurons inhibiting the release of neuropeptides and glutamate, which are responsible of nociception. Therefore, there is increasing evidence that the therapeutic effect in both motor and autonomic disorders is based on a complex mode of botulinum neurotoxin action modulating the activity of efferent as well as afferent nerve fibres.

The effect of coniine on presynaptic nicotinic receptors.

Toxicity of coniine, an alkaloid of Conium maculatum (poison hemlock), is manifested by characteristic nicotinic clinical signs including excitement, depression, hypermetria, seizures, opisthotonos via postsynaptic nicotinic receptors. There is limited knowledge about the role of presynaptic nicotinic receptors on the pharmacological and toxicological effects of coniine in the literature. The present study was undertaken to evaluate the possible role of presynaptic nicotinic receptors on the pharmacological and toxicological effects of coniine. For this purpose, the rat anococcygeus muscle and guinea-pig atria were used in vitro. Nicotine (100 μM) elicited a biphasic response composed of a relaxation followed by contraction through the activation of nitrergic and noradrenergic nerve terminals in the phenylephrine-contracted rat anococcygeus muscle. Coniine inhibited both the nitrergic and noradrenergic response in the muscle (-logIC(50) = 3.79 ± 0.11 and -logIC(50) = 4.57 ± 0.12 M, respectively). The effect of coniine on nicotinic receptor-mediated noradrenergic transmission was also evaluated in the guinea-pig atrium (-logIC(50) = 4.47 ± 0.12 M) and did not differ from the -logIC(50) value obtained in the rat anococcygeus muscle. This study demonstrated that coniine exerts inhibitory effects on nicotinic receptor-mediated nitrergic and noradrenergic transmitter response.

Abstract P067: Novel Antagonists of Dopamine-β-hydroxylase Identified and Validated Through Structure Based Approach to Combat Hypertension

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension, cardiac hypertrophy and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design was elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules UDSC171, UDSC180 and UDSC142 were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules were done in small model systems like C. elegans and D. melanogaster reconfirming their nontoxic properties up to 15μM doses. These three leads are now being tested in cardiac hypertrophy and hypertension rat models with exciting preliminary results.

Effects of various pharmacological agents on the function of norepinephrine transporter.

The norepinephrine transporter is selectively expressed in noradrenergic nerve terminals, where it can exert spatial and temporal control over the action of norepinephrine. The norepinephrine transporter mediates the termination of neurotransmission via the reuptake of norepinephrine released into the extracellular milieu. In the present brief review, we report our recent studies about the effects of various pharmacological agents such as fasudil, nicotine, pentazocine, ketamine and genistein on norepinephrine transporter function.

Effects of angiotensin type 2 receptor on secretion of the locus coeruleus in stress-induced hypertension rats.

Locus coeruleus (LC) has noradrenergic nerve terminals projecting to hypothalamus that modulating cardiovascular activity. To study the dynamic characteristics of norepinephrine (NE) release in hypothalamus followed by electrical stimulation in the locus coeruleus in the stress-induced hypertension (SIH) rats, we established the hypertension model rats by stimulations combining noise and foot-shock stress. After the end of modeling, NE release in the hypothalamus by electrical stimulation in LC was studied and NE signal was recorded by carbon fiber electrode. The peak value, the time to peak and half-life period of NE signal in both group rats were analyzed. Furthermore, to clarify the role of angiotensin II type 2 receptors (AT2) in norepinephrine (NE) release and the blood pressure of rat model of stress-induced hypertension, we intraperitoneally administered the AT2 receptor antagonist PD123319 (AT2 receptor antagonist, 0.3mg/kg, i.p.) and intracerebroventricularly injection of CGP42112 (AT2 receptor agonist, 6μg/5μl, i.c.v.) to adult male rats. We found the peak value of NE signal in the hypothalamus followed by electrical stimulation in the LC in SIH rats were higher than that in controls (P<0.01). Intraperitoneal injection of PD123319 (AT2 receptor antagonist) potentiated electrical stimulation in the LC induced NE release in the hypothalamus in SIH rats and elevated blood pressure (P<0.05), whereas intracerebroventricular injection of CGP42112 (AT2 receptor agonist) inhibited the NE release and reduced the heart rate (P<0.05). These results suggest that combining noise and foot-shock stresses increased the blood pressure and the secretion of NE in the hypothalamus followed by electrical stimulation in the LC in rats. AT2 receptors can inhibit the secretion of NE from the LC to the hypothalamus. The attenuation of presynaptic action of AT2 receptor may play a role in the pathophysiological mechanism of SIH in rats.

Abstract 267: Identification And Validation Of New Inhibitors Based On Rational Design Against Dopamine-β-hydroxylase To Combat Hypertension

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design has been elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules (NSC637578, NSC99657 and NSC379555) were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules has been initiated in model systems like C. elegans and D. melanogaster. These studies reconfirmed their nontoxic properties up to 15μM doses. CombiGlide and Ligand Based Core Hopping methods are being employed to optimize the lead compounds computationally to improve their pharmacokinetic properties.

DSP4, a selective neurotoxin for the locus coeruleus noradrenergic system. A review of its mode of action.

DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) is a selective neurotoxin for the locus coeruleus noradrenergic system in the rodent and bird brain. It readily passes the blood-brain barrier and cyclizes to a reactive aziridinium derivative that is accumulated into the noradrenergic nerve terminals via the noradrenaline transporter. DSP4 is also an irreversible inhibitor of this transporter. Within the nerve terminals the aziridinium derivative reacts with unknown vital cellular components, destroying the terminals. At the dose 50 mg/kg i.p. this is characterized by a rapid and long-lasting loss of noradrenaline and a slower decrease in the dopamine-β-hydroxylase enzyme activity and immunoreactivity in the regions innervated from locus coeruleus. The tissue level of noradrenaline is reduced to 10-30% of the normal value. The extraneuronal concentration is, on the other hand, increased due to inflow from non-lesioned regions. Like the peripheral sympathetic nerves the non-locus coeruleus noradrenergic systems in the rodent brain is resistant to the neurotoxic action of DSP4. Serotoninergic and dopaminergic nerves are only slightly or not at all affected by DSP4. The neurotoxic effect is counteracted by pretreatment with noradrenaline uptake inhibitors (e.g., desipramine). MAO-B inhibitors of the N-propargylamine type (e.g., selegiline) also counteract the DSP4-induced neurotoxicity with another, yet unknown mechanism. Because of its selectivity for the locus coeruleus system DSP4 is a useful tool in studies of the functional role of this noradrenergic system in the brain.

Involvement of the paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus in the central noradrenergic regulation of liver cytochrome P450

The present study was aimed at assessing the influence of noradrenergic innervation of the paraventricular nucleus (PVN) and the arcuate nucleus (ARC) of the brain hypothalamus on cytochrome P450 expression in the liver. DSP-4, a neurotoxin specific to noradrenergic nerve terminals, was administrated locally into the PVN or ARC. One week after neurotoxin injection, the levels of neurotansmitters (noradrenaline/dopamine/serotonin) were measured in the middle part of the hypothalamus, hormone concentrations were estimated in blood plasma, and the activity and the protein levels of CYP isoforms were measured in the liver. A significant decrease in noradrenaline level in the hypothalamus was observed after DSP-4 injection into the PVN or ARC. The levels of dopamine or serotonin remained unchanged or slightly lowered. Simultaneously, significant changes in the plasma concentration of growth hormone were found; its elevation in PVN-lesioned rats and a drop in ARC-lesioned ones. There were no changes in the plasma concentration of the thyroid hormones or corticosterone. The activity and protein levels of isoforms CYP2C11, CYP3A and CYP2A rose in the liver of PVN-lesioned rats, but the activity and protein level of CYP2C11 fell in ARC-lesioned animals such a tendency being also observed in the case of CYP3A. Our study shows that noradrenergic innervation of the PVN and ARC of the hypothalamus exerts an opposite effect on the regulation of cytochrome P450 in the liver. These findings may be important for pharmacological experiments and pharmacotherapy with neuroactive drugs, since cytochrome P450 is responsible for the metabolism of steroids and the majority of drugs.

Myocardial infarction increases noradrenergic activity in medial prefrontal cortex and impairs consolidation of fear extinction more in female rats

Anxiety often develops after myocardial infarction (MI), particularly in women. Patients with anxiety often show a deficit in the ability to recall that a fearful cue no longer predicts danger (extinction recall), a process that requires adrenergic receptor activation in the infralimbic medial prefrontal cortex (ILmPFC). Here, we assessed noradrenergic activity in the ILmPFC and extinction recall after MI. Rats were subjected to fear conditioning with footshock, and sham or real coronary artery ligation. 8 weeks after surgery, male MI rats (n=4) had larger fear responses to the conditioned cue compared to male controls (n=6, P&lt;0.05). Fear could be extinguished in both groups, and both readily recalled extinction 24 hrs later. Female MI rats (n=6) showed similar fear to the cue and fear extinction as female controls (n=6), but they did not recall extinction 24hrs later (P&lt;0.05). Tyrosine hydroxylase expression was increased in noradrenergic nerve terminals of the ILmPFC after MI (P&lt;0.05) and the effect was exaggerated in female rats (P&lt;0.05). We conclude that chronic increases in noradrenergic activity in the ILmPFC may contribute to deficits in consolidation of extinction memories after MI in females

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome

SUMMARYPostural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET+/P) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET+/+) mice, whereas transport activity in mice carrying two A457P alleles (NETP/P) was nearly abolished. NET+/P and NETP/P mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET+/P mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET+/P mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

The α1-, but not α2-, adrenoceptor in the nucleus accumbens plays an inhibitory role upon the accumbal noradrenaline and dopamine efflux of freely moving rats

In vivo microdialysis was used to analyse the role of the α1- and α2-adrenoceptor subtypes in the regulation of noradrenaline and dopamine efflux in the nucleus accumbens of freely moving rats. Intra-accumbal infusion of α1-adrenoceptor agonist methoxamine (24pmol) failed to alter the noradrenaline efflux, but decreased the dopamine efflux. The intra-accumbal infusion of α1-adrenoceptor antagonist prazosin (6, 600 and 6000pmol) produced a dose-related increase and decrease of the noradrenaline and dopamine efflux, respectively. An ineffective dose of prazosin (6pmol) counteracted the methoxamine (24pmol)-induced decrease of dopamine efflux. The prazosin (6000pmol)-induced increase of noradrenaline efflux, but not the decrease of dopamine efflux, was suppressed by the co-administration of an ineffective dose of methoxamine (0.024pmol). Neither the α2-adrenoceptor agonist clonidine (300pmol) and UK 14,304 (300pmol) nor the α2-adrenoceptor antagonist RX 821002 (0.6, 3, 600 and 6000pmol) significantly affected the accumbal noradrenaline and dopamine efflux. The doses mentioned are the total amount of drug over the 60-min infusion period. The present results show that (1) accumbal α1-adrenoceptors which are presynaptically located on noradrenergic nerve terminals inhibit the accumbal noradrenaline efflux, increasing thereby the accumbal dopamine efflux, (2) accumbal α1-adrenoceptors which are postsynaptically located on dopaminergic nerve terminals inhibit the accumbal dopamine efflux, and (3) accumbal α2-adrenoceptors play no major role in the regulation of accumbal efflux of noradrenaline and dopamine.

Effects of the noradrenergic neurotoxin DSP-4 on the expression of α1-adrenoceptor subtypes after antidepressant treatment

AbstractWe have previously reported that chronic imipramine and electroconvulsive treatments increase the α1A-adrenoceptor (but not the α1B subtype) mRNA level and the receptor density in the rat cerebral cortex. Furthermore, we have also shown that chronic treatment with citalopram does not affect the expression of either the α1A- or the α1B-adrenoceptor, indicating that the previously observed up-regulation of α1A-adrenoceptor may depend on the noradrenergic component of the pharmacological mechanism of action of these antidepressants. Here, we report that previous noradrenergic depletion with DSP-4 (50mg/kg) (a neurotoxin selective for the noradrenergic nerve terminals) significantly attenuated the increase of α1A-adrenoceptor mRNA induced by a 14-day treatment with imipramine (IMI, 20mg/kg, ip) and abolished the effect of electroconvulsive shock (ECS, 150mA, 0.5s) in the prefrontal cortex of the rat brain. The changes in the receptor protein expression (as reflected by its density) that were induced by IMI and ECS treatments were differently modulated by DSP-4 lesioning, and only the ECS-induced increase in α1A-adrenoceptor level was abolished. This study provides further evidence corroborating our initial hypothesis that the noradrenergic component of the action of antidepressant agents plays an essential role in the modulation of α1A-adrenoceptor in the rat cerebral cortex.

6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs.

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A ΔqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the ΔqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.

Neonatal DSP-4 treatment impairs 5-HT1B receptor reactivity in adult rats. Behavioral and biochemical studies

To examine the effect of a central noradrenergic lesion on the reactivity of the 5-HT1B receptor we compared intact male rats with rats in which noradrenergic nerve terminals were largely destroyed with the neurotoxin DSP-4 (50mg/kg×2, on the 1st and 3rd days of postnatal life). When rats attained 10 weeks of age, control and DSP-4 rats were divided into two subgroups receiving either saline or the serotonin (5-HT) synthesis inhibitor (p-chlorophenylalanine; p-CPA; 100mg/kg). Employing an elevated plus maze test, we demonstrated that CP 94,253 (5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine hydrochloride)(4.0mg/kg; 5-HT1B agonist) induced an anxiogenic-like action in control rats; however, it failed to elicit this effect in the DSP-4 group. Surprisingly, in p-CPApretreated rats anxiogenic-like activity was observed both in control and DSP-4 treated rats. CP 94,253 significantly attenuated 5-HT synthesis in the medial prefrontal cortex (mPFC) of control rats, and SB 216641 (N-{3-[3-(dimethyl-amino)ethoxy]-4-methoxyphenyl}-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1’-biphenyl]-4-carboxamide hydrochloride) (4.0mg/kg; 5-HT1B antagonist) was able to antagonize this effect. Conversely, CP 94,253 failed to significantly inhibit the 5-HT synthesis rate in DSP-4-treated rats. In the microdialysis study CP 94,253 induced long-lasting attenuation of 5-HT release in the mPFC of control rats but had no effect in DSP-4 rats. These data lead to the proposal that presynaptic 5-HT1B autoreceptors underwent desensitization in DSP-4 treated rats.

The Galectin-1 level in serum as a novel marker for stress

Galectin-1(Gal-1), a carbohydrate-binding protein with an affinity for beta-galactoside, is widely expressed in various normal and pathological tissues and it also plays an important role in regulating immune cell homeostasis and tumorigenesis. This study investigated the effects of restraint stress on serum Gal-1 by Western blot analyses and enzyme-linked immunosorbent assays. The Gal-1 levels of the restraint-stress group were significantly higher than those of the control group. However, this increase by stress was not obvious in adolescent rats. The pattern of these changes was similar to that of corticosterone. Furthermore, this Gal-1 increase in the serum was prevented by pre-treatment with a neurotoxin 6-hydroxydopamine (6-OHDA), which destroys the noradrenergic nerve terminals. However, a bilateral adrenalectomy (ADX) had no effect on the Gal-1 increase. These results suggest that Gal-1 is a candidate stress marker protein and that the stress-induced increase of Gal-1 in serum is regulated by the sympathetic nervous system under stress conditions.