Norepinephrine Transporter mRNA Levels Research Articles

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Norepinephrine transporter expressed on mammary epithelial cells incorporates norepinephrine in milk into the cells

Mammary epithelial cells synthesize and secrete norepinephrine (NE) into breast milk to regulate β-casein expression through the adrenergic β2 receptor. We investigated the expression, localization, and roles of NE transporter (NET) in the mammary epithelium during lactation. mRNA and protein levels of NET were determined in primary normal human mammary epithelial cells (pHMECs) and non-malignant human mammary epithelial MCF-12A cells. In nursing CD1 mice, NET localized to the apical membranes of the mammary epithelium. The intracellular NE content of pHMECs incubated with NE increased. Although the β-casein concentration in milk was slightly higher at day 10 than at day 2 of lactation, the NE concentration and lactation-related proteins were only slightly changed on days 2–10. Restraint stress increased the NE concentration in milk from nursing mice and NET protein levels were significantly higher than in non-stressed nursing mice. NET is expressed on the apical membrane of mammary epithelial cells and incorporates NE in milk into cells, potentially regulating the NE concentration in milk.

MicroRNAs 29b and 181a down-regulate the expression of the norepinephrine transporter and glucocorticoid receptors in PC12 cells.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation invitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

Corticotropin releasing factor up-regulates the expression and function of norepinephrine transporter in SK-N-BE (2) M17 cells.

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) invitro. 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 [(3) H] 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 invitro. 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.

Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine β-hydroxylase in rat locus coeruleus and its terminal regions.

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.

Effects of DSP4 on the Noradrenergic Phenotypes and Its Potential Molecular Mechanisms in SH-SY5Y Cells

Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.

Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli

Estrogen deficiency increases the risk of anxiety and mood disorders, presumably by deranging metabolism of the monoamine neurotransmitters and the expression of their reuptake transporters in the brain. Although estrogen-deficient individuals were also susceptible to stress, little was known regarding the effect of stress on the levels of transcripts related to brain monoamine metabolism. Herein, we used quantitative real-time PCR to quantify the mRNA levels of serotonin reuptake transporter (SERT), norepinephrine transporter (NET), monoamine oxidase-B (MAOB), tryptophan hydroxylase (TPH), and tyrosine hydroxylase (TH) in various brain regions of ovariectomized (OVX) rats which had been exposed for 4 weeks to chronic aversive stimuli (CAS), such as water deprivation, cage tilt, and illumination. We found that CAS induced stress responses in OVX rats as indicated by increases in the adrenal gland weight and sucrose intake. After CAS exposure, mRNA levels of SERT and NET were upregulated in the frontal cortex, hippocampus, amygdala, and periaqueductal gray. In addition, CAS also increased the mRNA levels of MAOB, an enzyme for dopamine degradation, in the same brain regions. However, CAS did not alter the mRNA levels of TPH or TH, both of which are rate-limiting enzymes for the synthesis of serotonin and norepinephrine in the dorsal raphé and locus coeruleus, respectively. Interestingly, mRNA expression of brain-derived neurotrophic factor precursor was upregulated in the hippocampus of CAS-exposed OVX rats, suggesting a compensatory mechanism which might counteract the stress-induced depression. Therefore, the present data have provided evidence to explain how stress affected brain monoamine metabolism in estrogen-deficient stressed patients.

NF‐κB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor-kappaB (NF-κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I-metaiodobenzylguanidine (131I-MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF-κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF-κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF-κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF-κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I-MIBG and by the induction of apoptosis.

Angiotensin‐(1–7) through Mas receptor up‐regulates neuronal norepinephrine transporter via Akt and Erk1/2‐dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.

Dexamethasone‐induced up‐regulation of the human norepinephrine transporter involves the glucocorticoid receptor and increased binding of C/EBP‐β to the proximal promoter of norepinephrine transporter

Previously, we have found glucocorticoids up-regulate norepinephrine (NE) transporter (NET) expression in vitro. However, the underlying transcriptional mechanism is poorly understood. In this study, the role of glucocorticoids on the transcriptional regulation of NET was investigated. Exposure of neuroblastoma SK-N-BE(2)M17 cells to dexamethasone (Dex) significantly increased NET mRNA and protein levels in a time- and dose-dependent manner. This effect was attenuated by glucocorticoid receptor (GR) antagonist mifepristone, suggesting that up-regulation of NET by Dex was mediated by the GR. In reporter gene assays, exposure of cells to Dex resulted in dose-dependent increases of luciferase activity that were also prevented by mifepristone. Serial deletions of the NET promoter delineated Dex-responsiveness to a -301 to -148 bp region containing a CCAAT/enhancer binding protein-β (C/EBP-β) response element. Co-immunoprecipitation experiments demonstrated that Dex treatment caused the interaction of the GR with C/EBP-β. Chromatin immunoprecipitation (ChIP) assay revealed that Dex exposure resulted in binding of both GR and C/EBP-β to the NET promoter. Further experiments showed that mutation of the C/EBP-β response element abrogated C/EBP-β- and GR-mediated transactivation of NET. These findings demonstrate that Dex-induced increase in NET expression is mediated by the GR via a non-conventional transcriptional mechanism involving interaction of C/EBP-β with a C/EBP-β response element.

Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine β-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Vorinostat Increases Expression of Functional Norepinephrine Transporter in Neuroblastoma In Vitro and In Vivo Model Systems

Histone deacetylase (HDAC) inhibition causes transcriptional activation or repression of several genes that in turn can influence the biodistribution of other chemotherapeutic agents. Here, we hypothesize that the combination of vorinostat, a HDAC inhibitor, with (131)I-meta-iodobenzylguanidine (MIBG) would lead to preferential accumulation of the latter in neuroblastoma (NB) tumors via increased expression of the human norepinephrine transporter (NET). In vitro and in vivo experiments examined the effect of vorinostat on the expression of NET, an uptake transporter for (131)I-MIBG. Human NB cell lines (Kelly and SH-SY-5Y) and NB1691-luc mouse xenografts were employed. The upregulated NET protein was characterized for its effect on (123)I-MIBG biodistribution. Preincubation of NB cell lines, Kelly, and SH-SY-5Y, with vorinostat caused dose-dependent increases in NET mRNA and protein levels. Accompanying this was a corresponding dose-dependent increase in MIBG uptake in NB cell lines. Four- and 2.5-fold increases were observed in Kelly and SH-SY-5Y cells, respectively, pretreated with vorinostat in comparison to untreated cells. Similarly, NB xenografts, created by intravenous tail vein injection of NB1691-luc, and harvested from nude mice livers treated with vorinostat (150 mg/kg i.p.) showed substantial increases in NET protein expression. Maximal effect of vorinostat pretreatment in NB xenografts on (123)I-MIBG biodistribution was observed in tumors that exhibited enhanced uptake in vorinostat-treated [0.062 ± 0.011 μCi/(mg tissue-dose injected)] vs. -untreated mice [0.022 ± 0.003 μCi/(mg tissue-dose injected); P < 0.05]. The results of our study provide preclinical evidence that vorinostat treatment can enhance NB therapy with (131)I-MIBG.

Corticosterone up‐regulates expression and function of norepinephrine transporter in SK‐N‐BE(2)C cells

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 [(3)H]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.

Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine β‐hydroxylase in SK‐N‐BE(2)C cells

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 beta-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 mug 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 [(3)H]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 [(3)H]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.

Expressions of cardiac sympathetic norepinephrine transporter and β1-adrenergic receptor decreased in aged rats

Evidence suggests that the deterioration of communication between the sympathetic nervous system and cardiovascular system always accompanies the aging of human and animals. Cardiac sympathetic norepinephrine (NE) transporter (NET) on presynaptic membrane is a predominant component to eliminate released NE in the synaptic cleft and maintains the sensitivity of the beta-adrenergic receptor (beta-AR). In the present study, we investigated NET and beta1-AR mRNA levels and sympathetic nerve density in cardiac sympathetic ganglion and left ventricular myocardium in 2- and 16-month-old rats with Northern blot analysis and immunohistochemistry. The expression levels of NET mRNA, NET protein and beta1-AR mRNA in the ganglia or myocardia of 16-month-old rats were markedly reduced by 67%, 26%, and 43%, respectively, in comparison with those in 2-month-old rats. Our results also show that aging induces a strong decrease of the catecholaminergic nerve fiber density.

Postinfarct sympathetic hyperactivity differentially stimulates expression of tyrosine hydroxylase and norepinephrine transporter

The balance between norepinephrine (NE) synthesis, release, and reuptake is disrupted after acute myocardial infarction, resulting in elevated extracellular NE. Stimulation of sympathetic neurons in vitro increases NE synthesis and the synthetic enzyme tyrosine hydroxylase (TH) to a greater extent than it increases NE reuptake and the NE transporter (NET), which removes NE from the extracellular space. We used TGR(ASrAOGEN) transgenic rats, which lack postinfarct sympathetic hyperactivity, to test the hypothesis that increased cardiac sympathetic nerve activity accounts for the imbalance in TH and NET expression in these neurons after myocardial infarction. TH and NET mRNA levels were identical in the stellate ganglia of unoperated TGR(ASrAOGEN) rats compared with Sprague Dawley (SD) controls, but the threefold increase in TH and twofold increase in NET mRNA seen in the stellate ganglia of SD rats 1 wk after ischemia-reperfusion was absent in TGR(ASrAOGEN) rats. Similarly, the increase in TH and NET protein observed in the base of the SD ventricle was absent in the base of the TGR (ASrAOGEN) ventricle. Neuronal TH content was depleted in the left ventricle of both genotypes, whereas NET was unchanged. Basal heart rate and cardiac function were similar in both genotypes, but TGR(ASrAOGEN) hearts were more sensitive to the beta-agonist dobutamine. Tyramine-induced release of endogenous NE generated similar changes in ventricular pressure and contractility in both genotypes, but postinfarct relaxation was enhanced in TGR(ASrAOGEN) hearts. These data support the hypothesis that postinfarct sympathetic hyperactivity is the major stimulus increasing TH and NET expression in cardiac neurons.

Norepinephrine transporter mRNA and protein are increased in left stellate from deoxycorticosterone‐acetate (DOCA)‐salt hypertensive rats

Reduced norepinephrine (NE) reuptake and NE transporter (NET) in the heart are associated with hypertension. Sympathetic neurons in the stellate ganglia innervate the heart and direct nearly all cardiac sympathetic activity. The purpose of this study was to compare the regulation of NET in bilateral stellate ganglia of normotensive and hypertensive rats (DOCA-salt). Total RNA was isolated from ganglia and RT-qPCR was performed to analyze NET mRNA levels. NET mRNA levels were 4.48 fold higher in hypertensive left stellate than in normotensive left stellate (p=.13, n=6–7). NET mRNA in the right stellate was unchanged with hypertension (p>.05, n=7–10). Additional ganglia were fixed and sectioned for immunohistochemistry with a NET antibody against a cytoplasmic loop of the protein. All neurons in both ganglia were immunoreative for NET. Staining for NET in left stellate neurons from hypertensive animals was significantly more intense than normotensive (p<0.05, n=4). NET staining intensity in right stellate neurons was not different in hypertension (p>0.05, n=4). Increased NET mRNA and protein levels in the left stellate ganglia may serve as a compensatory mechanism when NET function is reduced in the heart during hypertension. This study indicates separate regulatory control of NET between right and left stellate, and also between nerve cell bodies and terminals. Supported by P01HL70687.

P22PHOX KNOCKDOWN ATTENUATES NOREPINEPHRINE TRANSPORTER REDUCTION IN RESPONSE TO ENDOTHELIN‐1 IN PC12 CELLS

In DOCA-salt hypertension, superoxide anion (O2-·) levels are increased in sympathetic ganglion neurons as compared to normotensive controls; this increase is mediated in part by endothelin-1 (ET-1) via the activation of NADPH oxidase. Increased O2-· reduces norepinephrine uptake via norepinephrine transporter (NET). We tested the hypothesis that ET-1 reduces NET expression in PC12 cells via increases in O2-·, and this effect can be abolished by knockdown of p22phox, an indispensable component of the O2-·-generating NADPH oxidase. Undifferentiated PC12 cells were transfected with short hairpin RNA (shRNA) targeting p22phox. Cells with stable expression of p22phox shRNA (shRNA-p22) had reduced p22phox mRNA (71% knockdown) and protein (55% knockdown). Compared to wild type PC12 cells (WT), O2-· production induced by ET-1 was diminished in shRNA-p22. To assess ET-1 effects mediated by NADPH oxidase on NET, both shRNA-p22 and WT were exposed to ET-1 (100nM) for 30min to 24hrs. ET-1 reduced NET mRNA and protein levels in WT cells; the mRNA levels decreased over time and reached their minimum at 2 hrs (3.41 fold decrease, n=3), while protein levels remained reduced for 24 hrs (68% decrease, n=3). In shRNA-p22 cells, neither NET mRNA nor protein levels were changed by ET-1 treatment. These results indicate that the effects of ET-1 on O2-· production and NET expression in PC12 cells are p22phox -dependent.

Injection of Nerve Growth Factor Into Stellate Ganglia Improves Norepinephrine Reuptake Into Failing Hearts

An impairment of cardiac norepinephrine reuptake through the neuronal norepinephrine transporter promotes depletion of cardiac norepinephrine stores and local cardiac sympathetic activation in heart failure. Nerve growth factor regulates differentiation and survival of adult sympathetic cells and is decreased in failing hearts. We hypothesized that injection of nerve growth factor into stellate ganglia normalizes cardiac norepinephrine homeostasis in experimental heart failure. Rats with transverse aortic constriction characterized by heart failure, depleted cardiac norepinephrine stores, and impaired cardiac norepinephrine reuptake were used as an experimental model. Nerve growth factor (20 microg) or saline was directly injected into left stellate ganglia 4 weeks after transverse aortic constriction. Thirty-two hours after injection, determinants of cardiac norepinephrine homeostasis were measured. As compared with saline, nerve growth factor refilled depleted cardiac norepinephrine stores and improved cardiac [3H]-norepinephrine uptake into isolated perfused hearts of transverse aortic constricted rats. In addition, pharmacological blockade of the norepinephrine transporter led to a higher increase in the overflow of endogenous norepinephrine from hearts of nerve growth factor-injected than saline-injected transverse aortic constricted rats. Norepinephrine transporter mRNA levels and the density of cardiac sympathetic nerves were not changed. Thirty-two hours after nerve growth factor injection, echocardiography revealed an increase in fractional shortening as compared with 2 days before injection. In conclusion, nerve growth factor attenuates local cardiac sympathetic overdrive of hypertrophic hearts by improving cardiac norepinephrine reuptake and might represent a novel therapeutic principle in the treatment of heart failure.

Norepinephrine Transporter: A Candidate Gene for Initial Ethanol Sensitivity in Inbred Long-Sleep and Short-Sleep Mice

Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.