Norepinephrine Transporter Protein Expression Research Articles

Dexmedetomidine reduces inflammation in mice with acute pancreatitis by inhibiting NLRP3 inflammasome and sympathetic nerve activity

Purpose: To study the anti-inflammatory influence of dexmedetomidine (DEX) in mice with acute pancreatitis (AP), and to determine the underlying mechanism.Methods: A total of 75 healthy ICR male mice were randomly divided into control, mild acute pancreatitis (MAP), MAP+DEX, severe acute pancreatitis (SAP), and SAP+DEX groups, with 15 mice/group. Blood levels of inflammatory factors (TNF-α and IL-1β) and norepinephrine were assayed ineach group. Western blotting was used to assay the protein expressions of NLRP3 and norepinephrine transporter (NET) in the pancreatic tissue of each group.Results: The levels of inflammatory factors in the MAP+DEX group were markedly lower than those in the MAP group after 10 h of MAP induction (p < 0.01). Mice in MAP+DEX group had significantly lower expression of NLRP3 in pancreatic tissue, and significantly higher NET protein level, relative to the MAP mice. Following 10 h of SAP, concentrations of the inflammatory factors and the pancreatic expression of NLRP3 were lower in SAP+DEX-treated mice than in SAP mice, while NET protein was significantly higher in SAP mice (p < 0.01).Conclusion: DEX reduces the expressions of inflammation-related factors TNF-α and IL-1β, and inhibits inflammatory response in mice with AP via downregulation of NET protein expression via inhibition of NLRP3 and early sympathetic events.
 Keywords: Dexmedetomidine, NLRP3 inflammasome, Sympathetic nerve, Acute pancreatitis, Inflammatory response

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.

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.

Sepsis affects the expression of Syntaxin1A and Norepinephrine transporter in the myocardium

Activation of the sympathetic nervous system leads to an increase release of norepinephrine (NE) during sepsis. Re‐uptake of NE by the NET (NE Transporter) protein is the primary mechanism by which the biological effects of NE in the synapse are terminated. A SNARE protein, Syntaxin 1A plays a major role in presynaptic regulatory mechanism linking neurotransmitter secretion to neurotransmitter reuptake activity. However the role of Syntaxin 1A in the regulation of NE in septic heart is not known. Therefore, in the present study the effect of increased duration of sepsis on the expression of syntaxin1A and NET in heart was elucidated. Male Sprague‐Dawley rats (300–350g) were randomized into sham, 1‐, 3‐ and 7‐day sepsis groups. Sepsis and sham‐sepsis was induced using 200mg/kg cecal inoculum and 5% dextrose water i.p., respectively. Hearts were isolated to perform the RT‐PCR and immunoblot analyses for Syntaxin 1A and NET levels. Syntaxin 1A mRNA as well as protein expression increased significantly at 1‐day, while reduced at 3‐ and 7‐day post‐sepsis. NET gene expression was not altered significantly in septic animals but NET protein expression was decreased (61%) in 3‐day post‐sepsis group. We also observed increased NE levels (50% compared to sham) at 1‐day post‐sepsis. The data suggest that the elevation of Syntaxin 1A could be responsible for an increased level of myocardial NE at 1‐day post‐sepsis. Disruption of the transporter function at 3‐day post‐sepsis may lead to an extraneuronal accumulation of the previously released NE in the heart. (This work was supported by NHLBI # 66016).

Down-regulation of norepinephrine transporter expression on membrane surface induced by chronic administration of desipramine and the antagonism by co-administration of local anesthetics in mice

We have previously shown that chronic administration of the antidepressant desipramine, a norepinephrine transporter (NET) inhibitor to mice markedly enhanced convulsions induced by local anesthetics and that behavioral sensitization may be relevant to decreased [ 3H]norepinephrine uptake by the isolated hippocampus. The co-administration of local anesthetics with desipramine reversed the behavioral sensitization and down-regulation of NET function induced by desipramine. The present study aimed to elucidate whether chronic treatment with desipramine regulates the expression of NET protein examined in membrane fractions in various brain regions and whether co-administration of local anesthetics affects the desipramine-induced alteration of NET expression. Desipramine with or without local anesthetics was injected intraperitoneally once a day for 5 days. The animals were decapitated 48 h after the last administration of drugs and the whole cell fraction, membrane fraction and cell-surface protein fraction were prepared. [ 3H]nisoxetine binding was significantly reduced in the P2 fraction of the hippocampus by chronic administration of desipramine, and the reduction was overcome by co-administration of lidocaine with desipramine. Immunoreactive NET was detected by SDS-PAGE and immunoblotting in the murine hippocampus. NET protein expression in the whole cell fraction and membrane fraction was not affected by treatment with any drugs. However, administration of desipramine significantly reduced the amount of immunoreactive NET in the cell-surface protein fraction. This reduction was blocked by simultaneous injection of lidocaine, bupivacaine or tricaine. These results indicate that the NET down-regulation indicated by the reduction of [ 3H]nisoxetine binding was induced by administration of desipramine via decrease of NET localization on the cell surface. The antagonistic actions of local anesthetics against NET down-regulation by desipramine were related to alterations of the cell-surface localization of NET.

Influence of the polyol pathway on norepinephrine transporter reduction in diabetic cardiac sympathetic nerves: implications for heterogeneous accumulation of MIBG

Cardiac scintigraphic studies using 123I-labeled metaiodobenzylguanidine ([123I]MIBG) have demonstrated heterogeneous myocardial accumulation of MIBG in diabetes. The accumulation has been found to correlate with a heterogeneous decrease in the expression of norepinephrine transporter (NET). In diabetic peripheral nerve tissue, polyol pathways are activated and cause nerve dysfunction and degeneration. However, there has been little research on the polyol pathway and cardiac sympathetic nerves. Therefore, to assess the influence of the polyol pathway on cardiac sympathetic nervous function, we investigated the regional accumulation of MIBG and NET protein expression in diabetic model rats treated with aldose reductase inhibitor (ARI) for the blockade of polyol pathways. Rats were given a single intravenous injection of streptozotocin (n=76, STZ-D rats). Starting the day after STZ injection, ARI was administered daily to 42 of the rats for 4 weeks (ARI-D rats). To assess the cardiac sympathetic nervous function, [125I]MIBG autoradiographic experiments were carried out. Finally, NET protein expression was assessed with a saturation binding assay. The myocardial sorbitol concentration was significantly higher in STZ-D rats than in ARI-D rats. There was no heterogeneous accumulation of MIBG in ARI-D rats. There was a heterogeneous decrease of NET expression in STZ-D rats, but not in ARI-D or control rats. The gathered data indicate that the enhanced polyol pathway correlates with the decrease in regional cardiac sympathetic nervous function, and this impairment may lead to the reduction of NET protein in cardiac sympathetic nerves of the diabetic inferior wall.

Influence of the polyol pathway on norepinephrine transporter reduction in diabetic cardiac sympathetic nerves: implications for heterogeneous accumulation of MIBG

Cardiac scintigraphic studies using (123)I-labeled metaiodobenzylguanidine ([(123)I]MIBG) have demonstrated heterogeneous myocardial accumulation of MIBG in diabetes. The accumulation has been found to correlate with a heterogeneous decrease in the expression of norepinephrine transporter (NET). In diabetic peripheral nerve tissue, polyol pathways are activated and cause nerve dysfunction and degeneration. However, there has been little research on the polyol pathway and cardiac sympathetic nerves. Therefore, to assess the influence of the polyol pathway on cardiac sympathetic nervous function, we investigated the regional accumulation of MIBG and NET protein expression in diabetic model rats treated with aldose reductase inhibitor (ARI) for the blockade of polyol pathways. Rats were given a single intravenous injection of streptozotocin (n=76, STZ-D rats). Starting the day after STZ injection, ARI was administered daily to 42 of the rats for 4 weeks (ARI-D rats). To assess the cardiac sympathetic nervous function, [(125)I]MIBG autoradiographic experiments were carried out. Finally, NET protein expression was assessed with a saturation binding assay. The myocardial sorbitol concentration was significantly higher in STZ-D rats than in ARI-D rats. There was no heterogeneous accumulation of MIBG in ARI-D rats. There was a heterogeneous decrease of NET expression in STZ-D rats, but not in ARI-D or control rats. The gathered data indicate that the enhanced polyol pathway correlates with the decrease in regional cardiac sympathetic nervous function, and this impairment may lead to the reduction of NET protein in cardiac sympathetic nerves of the diabetic inferior wall.

Norepinephrine transporter immunoblotting and radioligand binding in cocaine abusers

The norepinephrine transporter (NET) is a membrane protein responsible for transporting extracellular norepinephrine. The cocaine and tricyclic antidepressant-sensitive NET belongs to a family of sodium and chloride coupled transporters that include the monoamines dopamine and serotonin and the amino acids GABA and glycine. The regional distribution of the NET has been defined by synaptosomal uptake of norepinephrine and by autoradiographic approaches in rodent and primate brain. However, the NET has not been well characterized in the human brain due to the overall low abundance of protein expressed in axon terminals. Recently, immunolocalization studies have been used to identify the regional distribution of the cytoplasmic NET epitope in rodent brain. We report here on the characteristics of drug interactions with the native NET protein in human postmortem brain. Antisera raised against a 17-amino acid peptide from the N-terminus of the hNET recognized an 80 kDa species in human cerebral cortex. Chronic exposure to cocaine upregulated NET protein expression and [ 3 H]nisoxetine binding sites in the insular cortex from brains of cocaine addicts. These results demonstrate that immunologic and radioligand binding approaches afford specific labeling of the native transport protein in postmortem human brain.

Perinatal cocaine exposure reduces myocardial norepinephrine transporter function in the neonatal rat

Norepinephrine transporter (NET) mediates the active removal of norepinephrine (NE) released from sympathetic nerve terminals via reuptake, and NET function and expression can be regulated by cocaine. NET expression and its regulation by cocaine in the developing sympathetic nervous system during early postnatal period, however, have not been examined. We quantified immunodetectable NET protein expression in the neonatal rat heart to examine the developmental pattern of myocardial NET during the first 2 weeks after birth. To assess sympathetic innervations, we simultaneously quantified the expression of myocardial tyrosine hydroxylase (TH). Timed pregnant rats received daily intragastric treatment with saline (CTL) or cocaine at 60 mg/kg (Coc) from Gestational Day 2 until parturition. After birth, nursing mothers continued to receive the same treatment. The expression of myocardial TH and NET in neonatal rats were then studied at 1 day (Postnatal Day 1, PD1), 7 days (PD7) or 14 days (PD14) of age. We observed a similar age-dependent increase in the expression for myocardial NET and TH during the first 2 weeks of postnatal life, in both CTL and Coc animals. While myocardial TH was significantly up-regulated following perinatal cocaine exposure, no significant change in immunodetectable myocardial NET protein was evident. To further examine whether NET function might be affected by perinatal cocaine exposure, we performed NE uptake in myocardial membranes from PD14 CTL and Coc rats. We found that NE uptake was reduced at PD14 in the cocaine-treated group. Our results indicate that myocardial NET and TH are both developmentally regulated. Furthermore, our results indicate that perinatal exposure to cocaine did not change NET protein expression but impaired myocardial NET function in the neonatal rat.

Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells.

Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1-500 microM) either alone or in combination of Cu(2+) sulfate (1 microM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [(3)H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 microM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu(2+), which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event.