Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Molecular basis of human noradrenaline transporter reuptake and inhibition.

Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types1. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane2. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.

Etanercept ameliorates chronic mild stress-induced depressive-like behavior in rats: Crosstalk between MAPK and STAT3 pathways and norepinephrine and serotonin transporters.

Depression is a serious medical illness characterized by persistent feelings of sadness, hopelessness, and lack of interest in daily activities. It can interfere with daily functioning and quality of life. Despite decades of research, the pathophysiology of depression remains incompletely understood. The correlation between depression and inflammation has recently attracted considerable attention. This study investigated the potential antidepressant effect of etanercept, a tumor necrosis factor-alpha (TNF-α) inhibitor, utilizing a chronic mild stress (CMS) model in rats. Male Wistar rats were divided into two groups; one following a non-stressed protocol and the other a stressed protocol for 5 weeks. From the beginning of the third week, rats were treated either with saline daily or with etanercept twice a week (0.3 mg/kg, i.p.) or with fluoxetine daily (10 mg/kg, i.p) as a reference. Etanercept exhibited comparable effects to those of fluoxetine in counteracting CMS-induced behavioral manifestation in the forced swimming and splash tests. Etanercept also restored serotonin and norepinephrine levels to control values in the prefrontal cortex (PFC). Moreover, the current study verified the antioxidant and anti-inflammatory effects of etanercept. Interestingly, etanercept halted the expression of both norepinephrine and serotonin transporters in stressed rats. This could be attributed to abrogation of the p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription 3 (STAT-3) pathways in the PFC. The findings of the present study contribute to the understanding of the potential of etanercept as an antidepressant and provide insights into the neurobiological mechanisms underlying its therapeutic effects.

Perturbations in risk/reward decision making and frontal cortical catecholamine regulation induced by mild traumatic brain injury.

Mild traumatic brain injury (mTBI) disrupts cognitive processes that influence risk taking behavior. Little is known regarding the effects of repetitive mild injury (rmTBI) or whether these outcomes are sex specific. Risk/reward decision making is mediated by the prefrontal cortex (PFC), which is densely innervated by catecholaminergic fibers. Aberrant PFC catecholamine activity has been documented following TBI and may underlie TBI-induced risky behavior. The present study characterized the effects of rmTBI on risk/reward decision making behavior and catecholamine transmitter regulatory proteins within the PFC. Rats were exposed to sham, single (smTBI), or three closed-head controlled cortical impact (CH-CCI) injuries and assessed for injury-induced effects on risk/reward decision making using a probabilistic discounting task (PDT). In the first week post-final surgery, mTBI increased risky choice preference. By the fourth week, males exhibited increased latencies to make risky choices following rmTBI, demonstrating a delayed effect on processing speed. When levels of tyrosine hydroxylase (TH) and the norepinephrine reuptake transporter (NET) were measured within subregions of the PFC, females exhibited dramatic increases of TH levels within the orbitofrontal cortex (OFC) following smTBI. However, both males and females demonstrated reduced levels of OFC NET following rmTBI. These results indicate the OFC is susceptible to catecholamine instability after rmTBI and suggests that not all areas of the PFC contribute equally to TBI-induced imbalances. Overall, the CH-CCI model of rmTBI has revealed time-dependent and sex-specific changes in risk/reward decision making and catecholamine regulation following repetitive mild head injuries.

Feasibility Study of Single-Photon Emission Computed Tomography with Iodine-123 Labeled Metaiodobenzylguanidine for Preclinical Evaluation of Labetalol as a ß-Adrenergic Receptor Blocker.

Among clinically used radiopharmaceuticals, iodine-123 labeled metaiodobenzylguanidine ([123I]mIBG) serves for diagnosing neuroendocrine tumors and obtaining images of myocardial sympathetic innervation. mIBG, a structural analogue of norepinephrine (NE), a neurotransmitter acting in peripheral and central nerves, follows a pathway similar to NE, transmitting signals through the NE transporter (NET) located at synaptic terminals. It moves through the body without decomposing, enabling noninvasive image evaluation. In this study, we aimed to quantify [123I]mIBG uptake in the adrenal glands using small animal single-photon emission computed tomography/computed tomography (SPECT/CT) images post [123I]mIBG administration. We investigated the possibility of assessing the effectiveness of ß-adrenergic receptor blockers by quantifying SPECT/CT images and biodistribution results to determine the degree of [123I]mIBG uptake in the adrenal glands treated with labetalol, a known ß-adrenergic receptor blocker. Upon intravenous administration of [123I]mIBG to mice, SPECT/CT images were acquired over time to confirm the in vivo distribution pattern, revealing a clear uptake in the adrenal glands. Labetalol inhibited the uptake of [123I]mIBG in cell lines expressing NET. A decrease in [123I]mIBG uptake in the adrenal glands was observed in the labetalol-treated group compared with the normal group through SPECT/CT imaging and biodistribution studies. These results demonstrate that SPECT/CT imaging with [123I]mIBG could be applicable for evaluating the preclinical efficacy of new antihypertensive drug candidates such as labetalol, a ß-adrenergic receptor blocker.

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Norepinephrine transporter and vesicular monoamine transporter 2 tumor expression as a predictor of response to 131 I-MIBG in patients with relapsed/refractory neuroblastoma.

BACKGROUND: Prior studies suggest that norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) mediate meta-iodobenzylguanidine (MIBG) uptake and retention in neuroblastoma tumors. We evaluated the relationship between NET and VMAT2 tumor expression and clinical response to 131 I-MIBG therapy in patients with neuroblastoma. METHODS: Immunohistochemistry (IHC) was used to evaluate NET and VMAT2 protein expression levels on archival tumor samples (obtained at diagnosis or relapse) from patients with relapsed or refractory neuroblastoma treated with 131 I-MIBG. A composite protein expression H-score was determined by multiplying a semi-quantitative intensity value (0-3+) by the percentage of tumor cells expressing the protein. RESULTS: Tumor samples and clinical data were available for 106 patients, of whom 28.3% had partial response (PR) or higher. NET H-score was not significantly associated with response (≥PR), though the percentage of tumor cells expressing NET was lower among responders (median 80% for ≥PR vs. 90% for

Preliminary Evaluation of 18F-Labeled Benzylguanidine Analogs as NET Tracers for Myocardial Infarction Diagnosis.

PURPOSE: Heart failure (HF) remains a major cause of late morbidity and mortality after myocardial infarction (MI). To date, no clinically established 18F-labeled sympathetic nerve PET tracers for monitoring myocardial infarction are available. Therefore, in this study, we synthesized a series of 18F-labeled benzyl guanidine analogs and evaluated their efficacy as cardiac neuronal norepinephrine transporter (NET) tracers for myocardial imaging. We also investigated the preliminary diagnostic capabilities of these tracers in myocardial infarction animal models, as well as the structure-activity relationship of these tracers. PROCEDURES: Three benzyl guanidine-NET tracers, including [18F]1, [18F]2, and [18F]3, were synthesized and evaluated in vivo as PET tracers in a myocardial infarction mouse model. [18F]LMI1195 was used as a positive control for the tracers. H&E staining of the isolated myocardial infarction heart tissue sections was performed to verify the efficacy of the selected PET tracer. RESULTS: Our data show that [18F]3 had a moderate decay corrected labeling yield (~10%) and high radiochemical purity (>95%) compared to other tracers. The uptake of [18F]3 in normal mouse hearts was 1.7±0.1%ID/cc at 1 h post-injection (p. i.), while it was 2.4±0.1, 2.6±0.9, and 2.1±0.4%ID/cc in the MI mouse hearts at 1, 2, and 3 days after surgery, respectively. Compared with [18F]LMI1195, [18F]3 had a better myocardial imaging effect in terms of the contrast between normal and MI hearts. The area of myocardial infarction shown by PET imaging corresponded well with the infarcted tissue demonstrated by H&E staining. CONCLUSIONS: With an obvious cardiac uptake contrast between normal mice and the myocardial infarction mouse model, [18F]3 appears to be a potential tool in the diagnosis of myocardial infarction. Therefore, it is necessary to conduct further structural modification studies on the chemical structure of [18F]3 to improve its in vivo stability and diagnostic detection ability to achieve reliable and practical imaging effects.

Adrenal functional imaging - which marker for which indication?

PURPOSE OF REVIEW: In recent years, a broad spectrum of molecular image biomarkers for assessment of adrenal functional imaging have penetrated the clinical arena. Those include positron emission tomography and single photon emission computed tomography radiotracers, which either target glucose transporter, CYP11B enzymes, C-X-C motif chemokine receptor 4, norepinephrine transporter or somatostatin receptors. We will provide an overview of key radiopharmaceuticals and determine their most relevant clinical applications, thereby providing a roadmap for the right image biomarker at the right time for the right patient. RECENT FINDINGS: Numerous radiotracers for assessment of adrenal incidentalomas ([ 18 F]FDG; [ 123 I]IMTO/IMAZA), ACC ([ 123 I]IMTO/IMAZA; [ 18 F]FDG; [ 68 Ga]PentixaFor), pheochromocytomas and paragangliomas ([ 123 I]mIBG; [ 18 F]flubrobenguane; [ 18 F]AF78; [ 68 Ga]DOTATOC/-TATE), or primary aldosteronism ([ 11 C]MTO, [ 68 Ga]PentixaFor) are currently available and have been extensively investigated in recent years. In addition, the field is currently evolving from adrenal functional imaging to a patient-centered adrenal theranostics approach, as some of those radiotracers can also be labeled with ß-emitters for therapeutic purposes. SUMMARY: The herein reviewed functional image biomarkers may not only allow to increase diagnostic accuracy for adrenal gland diseases but may also enable for achieving substantial antitumor effects in patients with adrenocortical carcinoma, pheochromocytoma or paraganglioma.

Organic cation transporter 3 mediates the non-norepinephrine transporter driven uptake of meta-[211At]astato-benzylguanidine.

INTRODUCTION: Meta-[211At]astato-benzylguanidine ([211At]MABG) accumulates in pheochromocytoma via norepinephrine transporter (NET) and leads to a strong antitumor effect, but it also distributed in normal tissues non-specifically. Meta-[131I]iodo-benzylguanidine ([131I]MIBG), an iodine-labeled analog of [211At]MABG, is known to be transported by not only NET but also organic cation transporter (OCT). The involvement of OCT in [211At]MABG uptake is still largely unknown. We investigated the involvement of OCT in the non-NET-driven uptake of [211At]MABG both in vitro and in vivo. METHODS: [123I]MIBG and [211At]MABG uptake was investigated in PC-12 (rat pheochromocytoma cell line), NIH/3T3 (mouse fibroblasts cell line), ACHN (human renal cancer cell line), and BxPC-3 (human pancreatic cancer cell line). Herein, we used desipramine and dl-norepinephrine to inhibit NET, and we used steroids (hydrocortisone and prednisolone) to inhibit OCT3. The [211At]MABG uptake in OCT3-knockdown cells established with OCT3-selective siRNA was also investigated. We investigated the biodistribution of [211At]MABG in PC-12 tumor-bearing mice after a preloading of phosphate-buffered saline (PBS) or hydrocortisone solution. RESULTS: The uptake of both [123I]MIBG and [211At]MABG was significantly inhibited by desipramine in PC-12 cells but not the other cell lines. The expression of OCT3 was relatively higher than those of the other OCT subtypes in ACHN and BxPC-3 cells. The expression of OCTs was not observed in NIH/3T3 cells. The uptake of both [123I]MIBG and [211At]MABG in ACHN and BxPC-3 cells was significantly inhibited by the steroid treatments. The [211At]MABG uptake was also reduced in OCT3-knockdown cells (p < 0.001). The radioactivity of [211At]MABG was significantly reduced in normal tissues by the preloading of hydrocortisone. In contrast, there was an increasing trend of [211At]MABG uptake in the PC-12 tumors. The tumor-to-normal tissue ratio was significantly increased by the preloading of hydrocortisone compared to that of PBS. CONCLUSION: Our results suggest that OCT3 is involved in non-NET-driven [211At]MABG uptake. The preloading of hydrocortisone selectively reduced [211At]MABG accumulation in normal organs in vivo. OCT3 inhibition may therefore be beneficial for a reduction of the radiation risk in healthy organs in the treatment of malignant pheochromocytomas.

New Directions in Treatment of Metastatic or Advanced Pheochromocytomas and Sympathetic Paragangliomas: an American, Contemporary, Pragmatic Approach.

PURPOSE OF REVIEW: Multiple therapies with novel mechanisms have been explored in clinical trials for the treatment of metastatic pheochromocytomas and paragangliomas. We review current and future therapies for this disease and provide guidance on how and when to prescribe them based on tumor progression, clinical manifestations, molecular features, and social factors. RECENT FINDINGS: Approximately 60-70% of metastatic pheochromocytomas and paragangliomas express the noradrenaline transporter in their cell membranes. High specific activity iodine-131 metaiodobenzylguanidine has been recently approved by the US Food and Drug Administration for the treatment of metastatic pheochromocytomas and paragangliomas that express the noradrenaline transporter, in patients aged ≥ 12 years. More than 90% of patients treated with this medication exhibit clinical benefits. However, other therapies with novel mechanisms of action are needed to help all patients with this disease. Treatment of metastatic pheochromocytomas and paragangliomas is recommended based on the severity of symptoms, the progression of the disease, and the patient's performance status. Currently available therapies include surgery; systemic chemotherapy with cyclophosphamide, vincristine, and dacarbazine, or with temozolomide; high specific activity iodine-131 metaiodobenzylguanidine; peptide receptor radionuclide therapy; immunotherapy; tyrosine kinase inhibitors; and hypoxia-inducible factor 2 alpha inhibitors. Financial and social factors such as health insurance coverage and disparities also impact current clinical practice in the USA.

Norepinephrine transporter analog benzylguanidine-conjugated nanoparticles for the delivery of paclitaxel in neuroblastoma.

Aim: We previously synthesized a polyethylene glycol-based norepinephrine transporter-targeted agent, BG-P-TAT, which has a benzylguanidine and a triazolyl-tetrac group. This targeted conjugate showed suppression of neuroblastoma tumor progression. In this study we aimed to synthesize nanoparticles to encapsulate the chemotherapeutic agent paclitaxel for targeting neuroblastoma tumors by using benzylguanidine so that it can compete with norepinephrine for uptake by neuroendocrine cells. Methods: Biocompatible poly(lactide-co-glycolic acid)-polyethylene glycol was chosen to prepare targeted nanoparticles for safe delivery of the chemotherapy agent paclitaxel. Result: Paclitaxel concentration was 60% higher in neuroblastoma tumors of mice treated with paclitaxel encapsulated in targeted nanoparticles than with non-targeted nanoparticles. Conclusion: These findings support the targeted delivery of paclitaxel as a chemotherapeutic agent for neuroblastoma.

Novel [18F]-Labeled Meta-Bromobenzylguanidine Derivatives: Potential Positron Emission Tomography Imaging Probes for the Norepinephrine Transporter.

To develop novel norepinephrine transporter (NET)-targeting positron emission tomography (PET) probes with optimal pharmacokinetic properties, a series of meta-bromobenzylguanidine derivatives was synthesized. 4-Fluorodiethoxyethane-3-bromobenzylguanidine (compound 12) showed relatively good affinity for the NET (IC50 = 1.00 ± 0.04 µM). The corresponding radiotracer 18F-12 was prepared in high radiochemical purity (>98%) via a three-step method. The in vitro cellular uptake results demonstrated that 18F-12 was specifically taken up by NET-expressing SK-N-SH cells by the uptake-1 mechanism. Biodistribution studies in mice showed that 18F-12 exhibited high cardiac uptake (10.45 ± 0.66 %ID/g at 5 min p.i. and 6.44 ± 0.40 %ID/g at 120 min p.i.), faster liver clearance, and a lower dose of absorbed radiation than [123I]-labeled meta-iodobenzylguanidine ([123I]MIBG). Small animal PET imaging confirmed the high heart-to-background ratio of 18F-12 and the uptake-1 mechanism specific for the NET in rats, indicating its potential as a promising PET radiotracer for cardiac sympathetic nerve imaging.

Discovery of dual targeting PEGylated BG-P1600-TAT to norepinephrine transporter (NET) and thyrointegrin αvß3 in the treatment of neuroblastoma.

Polymer-drug conjugates are growing in interest as novel anticancer agents for targeted cancer therapy. The aim of this study was to synthesize a poly(ethylene glycol) (PEG) conjugated anticancer drug for neuroblastoma, which is the most common extracranial solid tumor of childhood and the deadliest tumor of infancy. In our previous studies, we designed and synthesized a dual targeting agent using benzylguanidine (BG) conjugated with the high affinity thyrointegrin αvß3 antagonist TriAzole Tetraiodothyroacetic acid (TAT) via non-cleavable bonding to PEG400 to make BG-P400-TAT and its derivatives as agents against neuroblastoma. Here, we improved the pharmacodynamic properties and increased the solubility by changing the polymer length to 1600 molecular weight. The TAT group, which acts as an integrin αvß3 antagonist, and the BG group, which can be taken up by neuroblastoma cells through the norepinephrine transporter (NET) system, are conjugated to PEG1600 to make BG-PEG1600-TAT. The binding affinity of BG-PEG1600-TAT was 40-fold higher to integrin αvß3 versus BG-P400-TAT and was associated with greater anticancer activities against neuroblastoma cells (SK-N-F1 and SKNAS) implanted in SCID mice along with broad spectrum anti-angiogenesis activities versus the FDA approved anti-Vascular Endothelial Growth Factor (VEGF) monoclonal antibody Avastin (bevacizumab). In conclusion, our novel dual targeting of NET and αvß3 receptor antagonist, BG-P1600-TAT demonstrated broad spectrum anti-angiogenesis and anti-cancer activities in suppressing neuroblastoma tumor progression and metastasis. Thus, BG-PEG1600-TAT represents a potential clinical candidate for targeted therapy in neuroblastoma management.

Design, synthesis, and biological evaluation of novel bifunctional thyrointegrin antagonists for neuroblastoma.

Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma membrane integrin αvß3. Dual targeting of the NET and integrin αvß3 receptors using a Drug-Drug Conjugate (DDC) might provide effective treatment strategy in the fight against neuroblastoma and other neuroendocrine tumors. In this work, we synthesized three dual-targeting BG-P400-TAT derivatives, dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400-PAT containing di-iodobenzene, di-methoxybenzene, and piperazine groups, respectively. These derivatives utilize to norepinephrine transporter (NET) and the integrin αvß3 receptor to simultaneously modulate both targets based on evaluation in a neuroblastoma animal model using the neuroblastoma SK-N-F1 cell line. Among the three synthesized agents, the piperazine substituted BG-P400-PAT exhibited potent integrin αvß3 antagonism and reduced neuroblastoma tumor growth and cancer cell viability by >90%. In conclusion, BG-P400-PAT and derivatives represent a potential therapeutic approach in the management of neuroblastoma.

Understanding the Polypharmacological Profiles of Triple Reuptake Inhibitors by Molecular Simulation.

The triple reuptake inhibitors (TRIs) class is a class of effective inhibitors of human monoamine transporters (hMATs), which includes dopamine, norepinephrine, and serotonin transporters (hDATs, hNETs, and hSERTs). Due to the high degree of structural homology of the binding sites of those transporters, it is a great challenge to design potent TRIs with fine-tuned binding profiles. The molecular determinants responsible for the binding selectivity of TRIs to hDATs, hNETs, and hSERTs remain elusive. In this study, the solved X-ray crystallographic structure of hSERT in complex with escitalopram was used as a basis for modeling nine complexes of three representative TRIs (SEP225289, NS2359, and EB1020) bound to their corresponding targets. Molecular dynamics (MD) and effective post-trajectory analysis were performed to estimate the drug binding free energies and characterize the selective profiles of each TRI to hMATs. The common binding mode of studied TRIs to hMATs was revealed by hierarchical clustering analysis of the per-residue energy. Furthermore, the combined protein-ligand interaction fingerprint and residue energy contribution analysis indicated that several conserved and nonconserved "Warm Spots" such as S149, V328, and M427 in hDAT, F317, F323, and V325 in hNET and F335, F341, and V343 in hSERT were responsible for the TRI-binding selectivity. These findings provided important information for rational design of a single drug with better polypharmacological profiles through modulating multiple targets.

Assessment of Epinephrine and Norepinephrine in Gastric Carcinoma.

The aim of our study was to assess the sympathetic nervous system's involvement in the evolution of gastric carcinoma in patients by analyzing the mediators of this system (epinephrine and norepinephrine), as well as by analyzing the histological expression of the norepinephrine transporter (NET). We conducted an observational study including 91 patients diagnosed with gastric carcinoma and an additional 200 patients without cancer between November 2017 and October 2018. We set the primary endpoint as mortality from any cause in the first two years after enrolment in the study. The patients were monitored by a 24-h Holter electrocardiogram (ECG) to assess sympathetic or parasympathetic predominance. Blood was also collected from the patients to measure plasma free metanephrine (Meta) and normetanephrine (N-Meta), and tumor histological samples were collected for the analysis of NET expression. All of this was performed prior to the application of any antineoplastic therapy. Each patient was monitored for two years. We found higher heart rates in patients with gastric carcinoma than those without cancer. Regarding Meta and N-Meta, elevated levels were recorded in the patients with gastric carcinoma, correlating with the degree of tumor differentiation and other negative prognostic factors such as tumor invasion, lymph node metastasis, and distant metastases. Elevated Meta and N-Meta was also associated with a poor survival rate. All these data suggest that the predominance of the sympathetic nervous system's activity predicts increased gastric carcinoma severity.

123I metaiodobenzylguanidine (MIBG) uptake predicts early relapse of neuroblastoma using semi-quantitative SPECT/CT analysis.

OBJECTIVE: 123I metaiodobenzylguanidine (MIBG) scintigraphy is a useful tool for the diagnosis of neuroblastoma (NB). MIBG uptake is correlated with norepinephrine transporter expression; hence, it is expected that high-MIBG tumors would be more highly differentiated and have a better prognosis than those with lower expression. We have introduced a method of assessing MIBG accumulation semi-quantitatively using SPECT/CT fusion images. The purpose of this study was to evaluate the relationship of 123I MIBG uptake measured by semi-quantitative values of SPECT/CT and early relapse of NB. METHODS: We studied the cases of 11 patients (5 males and 6 females, age 5-65 months, median age 20 months) with histopathologically proven NB between April 2010 and March 2015. The early-relapse group was defined as patients who had relapsed within 3 years after the first 123I MIBG SPECT/CT exam. Other patients were classified as the delay-relapse group. Uptake of MIBG was evaluated using the count ratio of tumor and muscles. T/Mmax and T/Mmean were defined as follows: T/Mmax = max count of tumor/max count of muscle, T/Mmean = mean count of tumor/mean count of muscle. RESULTS: The average T/Mmean values of the early-relapse group and delay-relapse group were 2.65 ± 0.58 and 7.66 ± 2.68, respectively. The T/Mmean values of the early-relapse group were significantly lower than those of delay-relapse group (p < 0.05). The average T/Mmax of the early-relapse group and delay-relapse group were 8.86 ± 3.22 and 16.20 ± 1.97, respectively. There was no significant difference in T/Mmax values between the two groups. CONCLUSIONS: Low 123I MIBG uptake using semi-quantitative SPECT/CT analysis was correlated with early relapse of NB.

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 ß-casein expression through the adrenergic ß2 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 ß-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.