Norepinephrine Transporter mRNA 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.

Thrombocytopenia after meta-iodobenzylguanidine (MIBG) therapy in neuroblastoma patients may be caused by selective MIBG uptake via the serotonin transporter located on megakaryocytes

BackgroundThe therapeutic use of [131I]meta-iodobenzylguanidine ([131I]MIBG) is often accompanied by hematological toxicity, primarily consisting of severe and persistent thrombocytopenia. We hypothesize that this is caused by selective uptake of MIBG via the serotonin transporter (SERT) located on platelets and megakaryocytes. In this study, we have investigated whether in vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG and whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent this radiotoxic MIBG uptake.MethodsPeripheral blood CD34+ cells were differentiated to human megakaryocytic cells using a standardized culture protocol. Prior to [3H]serotonin and [125I]MIBG uptake experiments, the differentiation status of megakaryocyte cultures was assessed by flow cytometry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess SERT and NET (norepinephrine transporter) mRNA expression. On day 10 of differentiation, [3H]serotonin and [125I]MIBG uptake assays were conducted. Part of the samples were co-incubated with the SSRI citalopram to assess SERT-specific uptake. HEK293 cells transfected with SERT, NET, and empty vector served as controls.ResultsIn vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell culture batches from three different hematopoietic stem and progenitor cell donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10–7 M MIBG (range: 6.6 ± 1.0 to 11.2 ± 1.0 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1%—41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes and SERT-specific MIBG uptake was observed.ConclusionOur study demonstrates that human megakaryocytes cultured in vitro are capable of MIBG uptake. Moreover, the SSRI citalopram selectively inhibits MIBG uptake via the serotonin transporter. The concomitant administration of citalopram to neuroblastoma patients during [131I]MIBG therapy might be a promising strategy to prevent the onset of thrombocytopenia.

Responses of PKCε to cardiac overloads on myocardial sympathetic innervation and NET expression.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. PKC activation play an important regulatory role of cardiac function. The present study was performed to investigate whether there were differential activations of the PKCε and how the activation coupled with norepinephrine transporter (NET) surface expression, sympathetic innervation pattern and extracellular matrix remodeling in different cardiac hemodynamic overloads induced by abdominal aortic constriction or aortocaval fistula. At 8weeks after the operations, heart failure were induced, accompanied with myocardial hypertrophy, which was more pronounced in pressure overload (POL) than that of volume overload (VOL) rats, left ventricular dysfunction and increased plasma norepinephrine (NE). In POL rats there was an increase in myocardial collagen deposition, in contrast, the amount decreased in VOL as compared with the sham rats. POL remarkably upregulated PKCε membrane-cytosol ratio and downregulated NET membrane fraction, whereas, in VOL induced opposite changes. Accompanied with the PKCε activation, nerve sprouting, evidenced by myocardial GAP43 protein increased, and different nerve phenotypes were found, in POL tyrosine hydroxylase (TH) positive nerve density increased with NET and choline acetyltransferase (ChAT) immunoreactivity density decreased, in contrast, in VOL NET and ChAT increased, TH did not change. The overloads did not induce alteration of NET mRNA expression, but resulted in different myocardial β1-AR mRNA expression, in POL β1-AR mRNAwas significantly downregulated, while in VOL rats unaltered. Conclusion, the present results suggested that the different cardiac hemodynamic overload could differentially activate a common signaling, PKCε intermediate and thereby generate biological diversity.

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.

Abstract 1610: Development of meta-[211At]astatobenzylguanidine ([211At]MABG) as an alpha particle emitting systemic targeted radiotherapeutic for neuroblastoma

Abstract BACKGROUND Neuroblastoma (NB) is a radiosensitive malignancy and NB cells express the norepinephrine transporter (NET) enabling uptake of NET ligands. The majority of relapsed NBs remain sensitive to radiation therapy even after extensive therapy. The currently employed targeted radiotherapeutic [131I]MIBG is a highly active β -particle emitting NET ligand useful for cytoreduction of bulk tumor. However, [131I]MIBG does not target isolated tumor cells, and thus targeted radiotherapy with [211At]MABG (α-particle emitter with higher biological effectiveness) can address this critical problem due to the short path length that should lead to the killing of isolated tumor cells. METHODS 211At was synthesized using a bismuth target via the 209Bi(α,2n)211At reaction and used for solid-phase radiosynthesis of [211At]MABG. Cellular models were created by first determining NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines, and then creating isogenic pairs by forced overexpression of NET in 4 NB cell models. We then performed [211At]MABG uptake assays, as well as biodistribution experiments, using these models. We are defining the toxicity of [211At]MABG in mice via ongoing dose escalation and monitoring laboratory parameters every 3 weeks after dosing. A linear mixed effects model was used to determine the differences in the parameters between the treated groups and controls. Finally, clonogenic assays and therapeutic trials in mouse models comparing [131I]MIBG with [211At]MABG are ongoing. RESULTS We synthesized [211At]MABG (radiochemical yield of 25-50%, radiochemical purity > 99%) and showed NET-specific uptake in NB1691 cells. Specificity was demonstrated by competition assays with the blocking agent desipramine. NET-overexpressing cell lines showed 4-10 fold higher uptake of NET ligands than parental isogenic lines, and demonstrated tumor-specific [211At]MABG uptake in vivo with tumor-muscle ratios of 7.37. Toxicity studies have shown that doses of 10 and 25 uCi of [211At]MABG were well tolerated, but there was weight loss observed at higher doses (P = 0.01). There was also an expected trend towards thrombocytopenia (P = 0.06), but no detectable impact on white blood cell counts, (P = 0.74), hemoglobin (P = 0.41), bilirubin (P = 0.53) or creatinine (P = 0.10). Clonogenic assays show [211At]MABG to be potently cytotoxic (1000 fold higher than [131I]MIBG), and murine efficacy studies are ongoing. CONCLUSIONS We have synthesized 211At-MABG in quantities sufficient for our preclinical experiments, and will scale this up for clinical applications. The uptake and biodistribution of [211At]MABG is similar to the currently used radiotherapeutic [131I]MIBG, and to date there was no unanticipated toxicity. Preliminary results from clonogenic assays suggest that [211At]MABG may be an effective agent for salvage therapy for children with refractory/relapsed NB. Citation Format: Vandana Batra, Pietro Ranieri, Mehran Makvandi, Matthew Tsang, Catherine Hou, Yimei Li, Ganesan Vaidyanathan, Daniel A. Pryma, John M. Maris. Development of meta-[211At]astatobenzylguanidine ([211At]MABG) as an alpha particle emitting systemic targeted radiotherapeutic for neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1610. doi:10.1158/1538-7445.AM2015-1610

Life-long norepinephrine transporter (NET) knock-out leads to the increase in the NET mRNA in brain regions rich in norepinephrine terminals

These studies aimed to identify the genes differentially expressed in the frontal cortex of mice bearing a life-long norepinephrine transporter knock-out (NET-KO) and wild-type animals (WT).Differences in gene expression in the mouse frontal cortex were studied using a whole-genome microarray approach. Using an alternative approach, i.e. RT-PCR (reverse transcription polymerase chain reaction) with primers complementary to various exons of the NET gene, as well as TaqMan arrays, the level of mRNA encoding the NET in other brain regions of the NET-KO mice was also examined. The analyses revealed a group of 92 transcripts (27 genes) that differentiated the NET-KO mice from the WT mice.Surprisingly, the studies have shown that the mRNA encoding NET accumulated in the brain regions rich in norepinephrine nerve endings in the NET-KO mice. Because there is no other source of NET mRNA besides the noradrenergic terminals in the brain regions studied, these results might speak in favor of the presence of mRNA in axon terminals. RNA-Binding Protein Immunoprecipitation approach indicated that mRNA encoding NET was detected in the Ago2 protein/mRNA complex. In addition, the amount of Ago2 protein in the frontal cortex was significantly higher in NET-KO mice as compared with that of the WT animals.These results are important for further characterization of the NET-KO mice, which – besides other merits – might serve as a good model to study the fate of truncated mRNA in neurons.

Abstract B48: Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma

Abstract Background: Neuroblastoma (NB) is a radiosensitive malignancy accounting for 10% of childhood cancer mortality. NB cells frequently express the norepinephrine transporter (NET) providing a specific mechanism for uptake of NET-ligands. Meta-[131I]iodobenzylguanidine ([131I]MIBG) is a NET-ligand radiotherapeutic that shows single-agent response rates in refractory NB of 40-50%. However, due to the long path lengths of 131 I beta (β)-emission, and low biological effectiveness compared to alpha (α)-emitting radionuclides, [131I]MIBG is generally not curative, perhaps due to non-targeting of isolated circulating tumor cells. Here we report our efforts to optimize NET-targeted radiotherapy by developing relevant preclinical models of refractory NB for α-particle therapeutic [211At] MABG therapy. Methods: We first determined NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines using quantitative RT-PCR and western blotting. We then chose 5 lines with absent to intermediate levels of native NET expression (NB1691, SKNSH, IMR5, NLF and SKNBE2) for dual forced overexpression of human NET and luciferase cDNAs. We used [125I]MIBG for cell-based uptake assays in all isogenic pairs and biodistribution experiments in athymic mice bearing three separate NET-transduced xenografts (N=5 per cell line). These cell lines were also treated with [131I]MIBG and/or external beam radiation (XRT) followed by multi-log cytotoxicity assays. Therapeutic trials of [131I]MIBG (25 mCi/kg) in NB1691 subcutaneous xenograft and metastatic mouse models were also conducted. In parallel, [211At] MABG was synthesized by: (i) cyclotron-production of 211 At via 209 Bi(α,2n)211At reaction (ii) distillation of 211 At from the target, and (iii) solid phase no-carrier-added synthesis of [211At] MABG by radioastato-destannylation. [211At] MABG uptake studies were performed in isogenic NB cell lines. Results: Unlike primary human NBs, NET expression was low in the majority of 35 cell-lines studied (median normalized expression value = 0.145; range 0.000-1.005), but all transduced lines showed significant overexpression (0.860-1.107) comparable to human primary tumors. Transduced lines showed 4-10 fold higher uptake of [125I]MIBG than non-transduced isogenic parental cell lines in vitro, and demonstrated significant tumor-specific uptake and retention in vivo with tumor-muscle ratios ranging from 13.80 to 29.48. In vitro cytotoxicity experiments using [131I]MIBG showed NET-expressing cell lines to be more susceptible to treatment compared to non-NET expressing pairs (IC50 of 2.937nCi vs. 15.99 nCi). Treatment of mice bearing NB1691-NET xenografts with [131I]MIBG showed tumor growth delay (p=0.0065), but no significant impact on survival, likely due to de novo radioresistance (1200 cGy of XRT had no impact on NB1691 proliferation; IMR-05 showed 97% decreased cell viability). Lastly, we successfully synthesized [211At] MABG, with radiochemical yields of ∼20% and showed NET specific uptake of [211At] MABG into 1691 NET transfected cells. Conclusions: Development of targeted radiotherapy for neuroblastoma has been limited by the lack of preclinical models and alternative therapeutics. Our development of multiple isogenic pairs with varying NET expression, documentation of de novo radiation sensitivity, and the production of [211At] MABG, will allow for rapid assessment of targeted radiotherapeutic strategies (including combination approaches) to support clinical development of alpha-particle therapeutics in a childhood cancer. Citation Format: V Batra, AM Chacko, M Gagliardi, C Hou, J L. Mikitsh, R H. Freifelder, A Kachur, B C. LeGeyt, A Schmitz, L Toto, G Vaidyanathan, M R. Zalutsky, K K. Matthay, W A. Weiss, W C. Gustafson, D Pryma, J M. Maris. Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B48.

Regional changes in cardiac and stellate ganglion norepinephrine transporter in DOCA–salt hypertension

Uptake of norepinephrine via the neuronal norepinephrine transporter is reduced in the heart during deoxycorticosterone (DOCA)-salt hypertension. We hypothesized that this was due to reduced norepinephrine transporter mRNA and/or protein expression in the stellate ganglia and heart. After 4 weeks of DOCA-salt treatment there was no change in norepinephrine transporter mRNA in either the right or the left stellate ganglia from hypertensive rats (n=5-7, p>0.05). Norepinephrine transporter immunoreactivity in the left stellate ganglion was significantly increased (n=4, p<0.05) while the right stellate ganglion was unchanged (n=4, p>0.05). Whole heart norepinephrine content was significantly reduced in DOCA rats consistent with reduced uptake function; however, when norepinephrine was assessed by chamber, a significant decrease was noted only in the right atrium and right ventricle (n=6, p<0.05). Cardiac norepinephrine transport binding by chamber revealed that it was only reduced in the left atrium (n=5-7, p>0.05). Therefore, 1) contrary to our hypothesis reduced reuptake in the hypertensive heart is not exclusively due to an overall reduction in norepinephrine transporter mRNA or protein in the stellate ganglion or heart, and 2) norepinephrine transporter regulation occurs regionally in the heart and stellate ganglion in the hypertensive rat heart.

Exogenous nerve growth factor supplementation elevates myocardial immunoreactivity and attenuates cardiac remodeling in pressure-overload rats

It is postulated that supplementation of exogenous nerve growth factor (NGF) might mediate improvement of the cardiac sympathetic nerve function in heart failure (HF). Local intramuscular injection of NGF near the cardiac sympathetic ganglia could influence the innervation pattern, norepinephrine transporter (NET) gene expression, and improve the cardiac remodeling in experimental HF animals. In this study, we injected NGF into the scalenus medius muscles of Sprague-Dawley rats with abdominal aortic constriction (AC). The nerve innervated pattern, left ventricular morphology, and function following injection in rats with AC were investigated respectively by immunohistochemistry and echocardiography. Levels of mRNA expression of NET, growth associated protein 43 (GAP 43), NGF and its receptors TrkA and p75(NTR), and brain natriuretic peptide (BNP) were measured by real-time polymerase chain reaction. The results showed that myocardial NGF mRNA levels were comparable in rats with AC. Short-term supplementation of exogenous NGF raised the myocardial NGF immunoreactivity, but did not cause hyperinnervation and NET mRNA upregulation in the AC rats. Furthermore, myocardial TrkA mRNA was found to be remarkably decreased and p75(NTR) mRNA was increased. Myocardial TrkA downregulation may play a beneficial effect for avoiding the hyperinnervation, and it is reasonable to postulate that p75(NTR) can function as an NGF receptor in the absence of TrkA. Interestingly, local NGF administration into the neck muscles near the ganglia could attenuate cardiac remodeling and downregulate BNP mRNA. These results suggest that exogenous NGF can reach the target tissue along the axons anterogradely, and improve the cardiac remodeling.

Evaluation of Norepinephrine Transporter Expression and Metaiodobenzylguanidine Avidity in Neuroblastoma: A Report from the Children's Oncology Group

Purpose. 123I-metaiodobenzylguanidine (MIBG) is used for the diagnostic evaluation of neuroblastoma. We evaluated the relationship between norepinephrine transporter (NET) expression and clinical MIBG uptake. Methods. Quantitative reverse transcription PCR (N = 82) and immunohistochemistry (IHC; N = 61) were performed for neuroblastoma NET mRNA and protein expression and correlated with MIBG avidity on diagnostic scans. The correlation of NET expression with clinical features was also performed. Results. Median NET mRNA expression level for the 19 MIBG avid patients was 12.9% (range 1.6–73.7%) versus 5.9% (range 0.6–110.0%) for the 8 nonavid patients (P = 0.31). Median percent NET protein expression was 50% (range 0–100%) in MIBG avid patients compared to 10% (range 0–80%) in nonavid patients (P = 0.027). MYCN amplified tumors had lower NET protein expression compared to nonamplified tumors (10% versus 50%; P = 0.0002). Conclusions. NET protein expression in neuroblastoma correlates with MIBG avidity. MYCN amplified tumors have lower NET protein expression.

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.

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.

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.

Upregulation of myocardial syntaxin1A is associated with an early stage of polymicrobial sepsis

This study was designed to test whether increased sympathetic stimulation during polymicrobial sepsis modulates the profile of the syntaxin1A and norepinephrine transporter (NET) in the heart. Sepsis of mild and severe intensity was induced in male Sprague-Dawley rats (275-350 g) using the cecal inoculum (CI) and cecal ligation and puncture (CLP) methods, respectively. The heart samples were isolated from sham, 1, 3, and 7 day post-sepsis in the CI model and at 2 and 20 h post-sepsis in the CLP model. In the CI model, the plasma concentration of norepinephrine (NE) significantly increased at 1, 3, and 7 days post-CI compared to the sham group. The myocardial syntaxin1A mRNA and protein expression also significantly increased at 1 day post-CI compared to the sham group. However, the sepsis-induced increase in syntaxin1A returned to the baseline values at 3 and 7 days post-CI. The expressions of myocardial NET mRNA and protein were not altered at 1 day post-CI but significantly decreased at 3 days post-CI compared to the sham and 1 day post-CI groups. The immunohistochemical analyses revealed an increased localization of NET and syntaxin1A in the heart tissue sections of the 1 day post-CI group. In the CLP model of severe sepsis, the myocardial syntaxin1A mRNA protein expressions significantly increased at 2 h post-CLP, but remained unchanged at 20 h post-CLP compared to the sham group. In contrast, the myocardial expressions of NET mRNA and protein significantly decreased at both 2 and 20 h post-CLP compared to the sham group. It appears that during severe sepsis (CLP model), both the upregulation of syntaxin1A and the downregulation of NET contribute to increased concentrations of NE during the early and late stages of sepsis.

Association of Changes in Norepinephrine and Serotonin Transporter Expression with the Long-Term Behavioral Effects of Antidepressant Drugs

Previous work has shown that repeated desipramine treatment causes downregulation of the norepinephrine transporter (NET) and persistent antidepressant-like effects on behavior, ie effects observed 2 days after discontinuation of drug treatment when acute effects are minimized. The present study examined whether this mechanism generalizes to other antidepressants and also is evident for the serotonin transporter (SERT). Treatment of rats for 14 days with 20 mg/kg per day protriptyline or 7.5 mg/kg per day sertraline reduced NET and SERT expression, respectively, in cerebral cortex and hippocampus; these treatments also induced a persistent antidepressant-like effect on forced-swim behavior. Increased serotonergic neurotransmission likely mediated the behavioral effect of sertraline, as it was blocked by inhibition of serotonin synthesis with p-chlorophenylalanine; a parallel effect was observed previously for desipramine and noradrenergic neurotransmission. Treatment with 20 mg/kg per day reboxetine for 42, but not 14, days reduced NET expression; antidepressant-like effects on behavior were observed for both treatment durations. Treatment for 14 days with 70 mg/kg per day venlafaxine, which inhibits both the NET and SERT, or 10 mg/kg per day phenelzine, a monoamine oxidase inhibitor, produced antidepressant-like effects on behavior without altering NET or SERT expression. For all drugs tested, reductions of NET and SERT protein were not accompanied by reduced NET or SERT mRNA in locus coeruleus or dorsal raphe nucleus, respectively. Overall, the present results suggest an important, though not universal, role for NET and SERT regulation in the long-term behavioral effects of antidepressants. Understanding the mechanisms underlying transporter regulation in vivo may suggest novel targets for the development of antidepressant drugs.

Analysis of Cardiac Norepinephrine (NE) Transporter (NET) mRNA after removal of the stellate ganglia.

NET is an important protein in the regulation of catecholamines, particularly NE, and its dysfunction has been linked to certain cardiovascular diseases. To evaluate the contribution of the sympathetic innervation from the stellate ganglia as a source of NET in the heart, we compared by qPCR NET mRNA in the heart in control rats to rats after removal of stellate ganglia. NET mRNA was present in heart homogenate, with atria expressing more NET mRNA than ventricles (p&lt;0.05, n=4–6). Adult, male rats underwent bilateral stellate ganglionectomy (SGX) or sham surgery and hearts were assayed by chamber two weeks later. To confirm successful denervation, NE content was measured by HPLC and was reduced uniformly by 85.96% in all heart chambers (p&lt;0.001, n=4–5). Although NE was depleted, there was a significant increase in NET mRNA (p&lt;0.05, n=5) in the left atria while no differences were seen in any other chambers (p&gt;0.05, n=4–6). The presence of NET mRNA after SGX suggests that NET mRNA is present in non‐stellate sources and is regionally increased by removal of the sympathetic innervation. P01HL79687, AHA predoctoral fellowship (EW).

Differential expression of cardiac neurotrophic factors and sympathetic nerve ending abnormalities within the failing heart

In congestive heart failure (CHF), cardiac sympathetic nerve endings transdifferentiate from a balanced norepinephrine (NE) storage/release/uptake apparatus to a nerve that predominantly releases NE. Little is known about the neurotrophic factors that may trigger this process. In the present study, we evaluated the cardiac expression pattern of nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in salt-sensitive Dahl rats (DS), which are characterized by profound alterations of the cardiac sympathetic nervous system. Experiments were performed in male DS and salt-resistant Dahl rats (DR) 30, 40 and 50 days after onset of high-salt intake. The sympathetic nerve density was measured by glyoxylic acid-induced histofluorescence. Cardiac NE re-uptake was assessed by isolated heart perfusion with [3H]-NE and norepinephrine transporter (NET) mRNA by real-time PCR. Cardiac expression of neurotrophic factors was determined by ribonuclease protection assay and Western blot analysis. DS rats displayed reduced left ventricular sympathetic nerve endings 40 days after onset of high-salt intake, which was preceded by an impaired cardiac [3H]-NE uptake. NGF, a positive regulator of NE re-uptake, and NT-3 were down-regulated already 30 days after onset of high-salt intake, whereas BDNF and CNTF protein expression were increased not before 40 days after onset of high-salt intake. In conclusion, during the development of CHF, a dysregulated NE storage/release/uptake apparatus within the sympathetic nerve endings might be triggered by differential expression of cardiac neurotrophic factors.