Structure Modeling of the Norepinephrine Transporter.

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds.

Regulation of monoamine transporters and receptors by lipid microdomains: implications for depression.

Lipid microdomains ("rafts") are dynamic, nanoscale regions of the plasma membrane enriched in cholesterol and glycosphingolipids, that possess distinctive physicochemical properties including higher order than the surrounding membrane. Lipid microdomain integrity is thought to affect neurotransmitter signaling by regulating membrane-bound protein signaling. Among the proteins potentially affected are monoaminergic receptors and transporters. As dysfunction of monoaminergic neurotransmission is implicated in major depressive disorder and other neuropsychiatric conditions, interactions with lipid microdomains may be of clinical importance. This systematic review evaluates what is known about the molecular relationships of monoamine transporter and receptor regulation to lipid microdomains. The PubMed/MeSH database was searched for original studies published in English through August 2017 concerning relationships between lipid microdomains and serotonin, dopamine and norepinephrine transporters and receptors. Fifty-seven publications were identified and assessed. Strong evidence implicates lipid microdomains in the regulation of serotonin and norepinephrine transporters; serotonin 1A, 2A, 3A, and 7A receptors; and dopamine D1 and ß2 adrenergic receptors. Results were conflicting or more complex regarding lipid microdomain associations with the dopamine transporter, D2, D3, and D5 receptors; and negative with respect to ß1 adrenergic receptors. Indirect evidence suggests that antidepressants, lipid-lowering drugs, and polyunsaturated fatty acids may exert effects on depression and suicide by altering the lipid milieu, thereby affecting monoaminergic transporter and receptor signaling. The lipid composition of membrane subdomains is involved in localization and trafficking of specific monoaminergic receptors and transporters. Elucidating precise mechanisms whereby lipid microdomains modulate monoamine neurotransmission in clinical contexts can have critical implications for pharmacotherapeutic targeting.

The association between in vivo central noradrenaline transporter availability and trait impulsivity.

The brain noradrenaline (NA) system, particularly NA transporters (NAT), are thought to play an important role in modulating impulsive behavior. Impaired impulsivity is implicated in a variety of neuropsychiatric conditions; however, an in vivo link between central NAT availability and human impulsivity has not been shown. Using positron emission tomography (PET) and S,S-[11C]O-methylreboxetine (MRB), we tested whether NAT availability is associated with this basic behavioral trait based on the Barratt Impulsiveness Scale (BIS-11) in twenty healthy individuals (12 females, 33.8±9.3, 21-52 years of age) with a body mass index (BMI) ranging from 21.7kg/m2 to 47.8kg/m2. Applying both voxel-wise and volume-of-interest (VOI) based analyses, we found that distribution volume ratios (DVR) used as PET outcome measures negatively correlated with BIS-11 total scores in the orbitofrontal cortex (OFC) and in the hippocampus as well as in parts of the cerebellar cortex. These associations however did not remain after correction for multiple testing. Thus, although it appears that low NAT availability is associated with greater scores of impaired behavioral control, this needs to be confirmed in a larger series of individuals with highly impulsive behavior.

Norepinephrine Transporter in Major Depressive Disorder: A PET Study.

OBJECTIVE: The norepinephrine transporter has been suggested to play a crucial role in major depressive disorder. However, norepinephrine transporter availability in major depressive disorder and its role with clinical symptoms are not known. The authors tested norepinephrine transporter availability in patients with major depressive disorder with the aim to identify any associations between test results and clinical symptoms. METHOD: The present research was a cross-sectional study in which 19 patients with major depressive disorder and 19 age- and sex-matched healthy comparison subjects underwent positron emission tomography scanning to evaluate the norepinephrine transporter availability measured by the radioligand (S,S)-[18F]FMeNER-D2. Norepinephrine transporter availability in the thalamus and its subregions was quantified in terms of nondisplaceable binding potential (BPND). The authors also analyzed the association between norepinephrine transporter availability and clinical symptoms. RESULTS: Compared with healthy subjects, patients with major depressive disorder showed 29.0% higher BPND values in the thalamus and, in particular, 28.2% higher values in the thalamic subregion anatomically connected to the prefrontal cortex. Elevated norepinephrine transporter availability in the thalamus in patients was positively correlated with attention, as measured by the Trail Making Test, part A. CONCLUSIONS: These findings revealed altered norepinephrine transmission in patients with major depressive disorder, suggesting that this alteration could be related to attention in this patient population.

Gestational stress, placental norepinephrine transporter and offspring fertility.

Chronic cold stress produces adrenergic overload that can affect fetal development. The placental norepinephrine transporter (NET) clears norepinephrine (NE) from both maternal circulation and the fetus during gestation. If this system fails, NE clearance can be reduced, leading to high fetal exposure to NE. The main aim of this study was to determine the changes in NET expression during gestation and their relationship with the functional capacity of NET to transport NE under stressful conditions. Additionally, this study correlated these findings with the reproductive capacity of 2nd-generation progeny. Pregnant rats were subjected to chronic cold stress at 4°C for 3 h each day throughout their pregnancies. We found that exposure of pregnant rats to sympathetic stress caused the following effects: increased NE and corticosterone levels throughout pregnancy, decreased capacity of the placenta to clear NE from the fetus to the mother's circulation, altered NET protein levels depending on the sex of the fetus and increased placental and body weights of pups. For the first time, we also described the disrupted fertility of progeny as adults. Increased NE plasma levels during pregnancy under sympathetic stress conditions correlated with decreased NET functionality that provoked changes in the development of progeny and their fertility in adulthood.

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

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

µ-Opioid receptor activation and noradrenaline transport inhibition by tapentadol in rat single locus coeruleus neurons.

BACKGROUND AND PURPOSE: Tapentadol is a novel analgesic that combines moderate µ-opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule. Both mechanisms of action are involved in producing analgesia; however, the potency and efficacy of tapentadol in individual neurons has not been characterized. EXPERIMENTAL APPROACH: Whole-cell patch-clamp recordings of G-protein-coupled inwardly rectifying K(+) (KIR 3.x) currents were made from rat locus coeruleus neurons in brain slices to investigate the potency and relative efficacy of tapentadol and compare its intrinsic activity with other clinically used opioids. KEY RESULTS: Tapentadol showed agonist activity at µ receptors and was approximately six times less potent than morphine with respect to KIR 3.x current modulation. The intrinsic activity of tapentadol was lower than [Met]enkephalin, morphine and oxycodone, but higher than buprenorphine and pentazocine. Tapentadol inhibited the noradrenaline transporter (NAT) with potency similar to that at µ receptors. The interaction between these two mechanisms of action was additive in individual LC neurons. CONCLUSIONS AND IMPLICATIONS: Tapentadol displays similar potency for both µ receptor activation and NAT inhibition in functioning neurons. The intrinsic activity of tapentadol at the µ receptor lies between that of buprenorphine and oxycodone, potentially explaining the favourable profile of side effects, related to µ receptors. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity.

The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [(3)H]noradrenaline uptake, [(3)H]nisoxetine binding and [(3)H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [(3)H]nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [(3)H]noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [(3)H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [(3)H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT.

The lignan (-)-hinokinin displays modulatory effects on human monoamine and GABA transporter activities.

The neurotransmitter transporters of the SLC6 family play critical roles in the regulation of neurotransmission and are the primary targets of therapeutic agents used to treat clinical disorders involving compromised neurotransmitter signaling. The dopamine and norepinephrine transporters have been implicated in clinical disorders such as attention deficit hyperactivity disorder (ADHD) and substance abuse. The GABA transporters (GATs) serve as a target for anxiolytic, antidepressant, and antiepileptic therapies. In this work, the interaction with neurotransmitter transporters was characterized for a derivative of the lignan (-)-cubebin (1), namely, (-)-hinokinin (2). Using in vitro pharmacological assays, 2 selectively inhibited the human dopamine and norepinephrine transporters, in a noncompetitive manner possibly mediated by binding to a novel site within the transporters, and displayed low affinity for the serotonin transporter. Compound 2 also specifically inhibited the GAT-1 GABA transporter subtype. Compound 2 is not a substrate of the carriers as it had no effect on the efflux of either of the neurotransmitters investigated. This compound is inactive toward glutamate and glycine transporters. These results suggest that 2 may serve as a tool to develop new therapeutic drugs for ADHD and anxiety that target the DAT, NET, and GAT-1 transporters.

Norepinephrine transporter function and human cardiovascular disease.

Approximately 80-90% of the norepinephrine released in the brain or in peripheral tissues is taken up again through the neuronal norepinephrine transporter (NET). Pharmacological studies with NET inhibitors showed that NET has opposing effects on cardiovascular sympathetic regulation in the brain and in the periphery. Furthermore, NET is involved in the distribution of sympathetic activity between vasculature, heart, and kidney. Genetic NET dysfunction is a rare cause of the postural tachycardia syndrome. The condition is characterized by excessive adrenergic stimulation of the heart, particularly with standing. Conversely, NET inhibition may be beneficial in hypoadrenergic states, such as central autonomic failure or neurally mediated syncope, which results from acute sympathetic withdrawal. Biochemical studies suggested reduced NET function in some patients with essential hypertension. Furthermore, cardiac NET function appears to be reduced in common heart diseases, such as congestive heart failure, ischemic heart disease, and stress-induced cardiomyopathy. Whether NET dysfunction is a consequence or cause of progressive heart disease in human subjects requires further study. However, studies with the nonselective NET inhibitor sibutramine suggest that reduced NET function could have an adverse effect on the cardiovascular system. Given the widespread use of medications inhibiting NET, the issue deserves more attention.

Inhibition of poly(ADP-Ribose) polymerase enhances the toxicity of 131I-metaiodobenzylguanidine/topotecan combination therapy to cells and xenografts that express the noradrenaline transporter.

UNLABELLED: Targeted radiotherapy using (131)I-metaiodobenzylguanidine ((131)I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining (131)I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)-expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance (131)I-MIBG/topotecan efficacy. METHODS: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and (131)I-MIBG and of PJ34 and (131)I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. RESULTS: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and (131)I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and (131)I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and (131)I-MIBG/topotecan combination treatment induced G(2) arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and (131)I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with (131)I-MIBG/topotecan therapy. CONCLUSION: The antitumor efficacy of topotecan, (131)I-MIBG, and (131)I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

Chronic mild stress-induced depression-like symptoms in rats and abnormalities in catecholamine uptake in small arteries.

OBJECTIVE: Major depression and cardiovascular diseases have a strong comorbidity; however, the reason for this is unknown. In the chronic mild stress (CMS) model of depression, only a fraction of rats develop a major feature of depression-anhedonia-like behavior, whereas other rats are stress resilient. Previous studies suggested that CMS rats also have increased total peripheral vascular resistance. METHODS: On the basis of CMS-induced changes of sucrose intake, a reliable measure for anhedonia, rats were divided into "resilient" and "anhedonic" groups. An interaction between hedonic status and vascular function was studied after 4 and 8 weeks of CMS exposure in vitro in wire myograph on saphenous arteries and mesenteric small arteries (MSAs) from these rats. RESULTS: When comparing the different experimental rat groups, arterial sensitivities to noradrenaline (NA) were similar under control conditions, but in the presence of the neuronal reuptake inhibitor cocaine, arteries from anhedonic rats were more sensitive to NA. No change in perivascular innervation was found, but elevated expression of neuronal NA transporter was detected. Inhibition of extraneuronal uptake with corticosterone (1 µM) suggests that this transport is diminished in MSAs after CMS. The corticosterone-sensitive transporter organic cation cotransporter 2 was shown to be reduced in MSAs after CMS. No CMS-induced changes in the corticosterone-sensitive transport were found in saphenous arteries. CONCLUSIONS: Our results indicate that CMS-induced depression-like symptoms in rats are associated with changes in catecholamine uptake pathways in the vascular wall, which potentially modulates the effect of sympathetic innervation of resistance arteries.

Dopamine in the hippocampus is cleared by the norepinephrine transporter.

Abnormal dopaminergic neurotransmission in the hippocampus may be involved in certain aspects of cognitive dysfunction. In the hippocampus, there is little, if any, expression of dopamine transporters (DAT), indicating that the mechanism for dopamine clearance differs from that in the striatum. Here, by means of in-vivo microdialysis in freely moving rats, we tested the hypothesis that the norepinephrine transporter (NET) is involved in dopamine clearance in the hippocampus. We found that systemic administration of the selective NET inhibitor reboxetine (3 mg/kg) and the psychostimulants amphetamine (0.5 mg/kg) and cocaine (10 mg/kg) increased hippocampal dopamine efflux. Local administration of reboxetine (300 µM) produced a large increase in hippocampal dopamine levels that could not be further enhanced by the addition of the NET/DAT inhibitor nomifensine (100 µM). Administration of the specific DAT inhibitor GBR12909 at a concentration (1 mM) that robustly increased dopamine in the nucleus accumbens had a comparably smaller effect in the hippocampus. In line with a minor role of DAT in the hippocampus, we detected very little DAT in this area using ligand binding with radiolabelled RTI-55. Moreover, in contrast to raclopride (100 µM), a dopamine D2-autoreceptor antagonist, local administration of the α2-adrenoceptor antagonist idazoxan (100 µM) increased hippocampal dopamine. Taken together, our data demonstrate an interaction between dopamine and norepinephrine systems in the hippocampus. It is proposed that this interaction originates from a shared uptake mechanism at the NET level.

Norepinephrine transporter (NET) knock-out upregulates dopamine and serotonin transporters in the mouse brain.

The noradrenaline, serotonin and dopamine transporters are three main transporters, which are the target of the antidepressant drugs. In the present study we demonstrate that the life-long deletion of the noradrenaline transporter (NET) induced up-regulation of two other monoamine transporters, dopamine and serotonin (DAT and SERT, respectively). An increase in the binding of [(3)H]paroxetine to the SERT and [(3)H]GBR12935 to the DAT was observed in various brain regions of NET-KO mice, without alterations of mRNA encoding these transporters, as measured by in situ hybridization. This important finding impacts the interpretation of previous data indicating the supersensitizity of NET-KO mice for psychostimulants or stronger effect of citalopram in behavioral tests. While using the NET-KO mice in various psychopharmacological studies is very important, one has to be aware that these mice lack NET from the earliest period of their existence, thus compensatory alterations do take place and have to be considered when it comes to interpretation of the obtained results.

The 1287 G/A polymorphism of the norepinephrine transporter gene (NET) is involved in commission errors in Korean children with attention deficit hyperactivity disorder.

BACKGROUND: Previous evidence supports the role of noradrenergic systems in ADHD, and norepinephrine transporter (NET) is critical in regulating the noradrenergic system. The present study aimed to investigate the association between NET gene polymorphism and the performance measures of the Continuous Performance Test (CPT) in Korean ADHD children. METHODS: Eighty-seven children (mean age = 9.23 ± 1.99 years) with ADHD were recruited from a university hospital. Genotypes of G1287A of the NET gene (SLC6A2) were analyzed. All participants completed the CPT, with performance measures of omission errors, commission errors, reaction time and reaction standardization computed. The relationship between G1287A polymorphisms and CPT performance measures was examined. RESULTS: There were 46 subjects with the G/G genotype, 35 subjects with the G/A genotype and 6 subjects with the A/A genotype. Among the three groups, there were no significant differences in the performance of CPTs. When dichotomized according to whether the subjects have the rare allele or not, subjects with the homozygous G/G genotype showed significantly lower commission errors compared to those without G/G genotypes (by independent T-test, t = -2.18, p = 0.026). DISCUSSION: Our study found a significant association between commission errors of the CPT and the G1287A genotype of the NET gene in Korean ADHD children. These findings suggest a protective role of the G/G genotype of the NET polymorphisms in the deficits of response inhibition in ADHD children.

Site-directed mutations near transmembrane domain 1 alter conformation and function of norepinephrine and dopamine transporters.

The human dopamine and norepinephrine transporters (hDAT and hNET, respectively) control neurotransmitter levels within the synaptic cleft and are the site of action for amphetamine (AMPH) and cocaine. We investigated the role of a threonine residue within the highly conserved and putative phosphorylation sequence RETW, located just before transmembrane domain 1, in regulating hNET and hDAT function. The Thr residue was mutated to either alanine or aspartate. Similar to the inward facing T62D-hDAT, T58D-hNET demonstrated reduced [(3)H]DA uptake but enhanced basal DA efflux compared with hNET with no further effect of AMPH. The mutations had profound effects on substrate function and binding. The potency of substrates to inhibit [(3)H]DA uptake and compete with radioligand binding was increased in T→A and/or T→D mutants. Substrates, but not inhibitors, demonstrated temperature-sensitive effects of binding. Neither the functional nor the binding potency for hNET blockers was altered from wild type in hNET mutants. There was, however, a significant reduction in potency for cocaine and benztropine to inhibit [(3)H]DA uptake in T62D-hDAT compared with hDAT. The potency of these drugs to inhibit [(3)H](-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) binding was not increased, demonstrating a discordance between functional and binding site effects. Taken together, these results concur with the notion that the T→D mutation in RETW alters the preferred conformation of both hNET and hDAT to favor one that is more inward facing. Although substrate activity and binding are primarily altered in this conformation, the function of inhibitors with distinct structural characteristics may also be affected.

A greater role for the norepinephrine transporter than the serotonin transporter in murine nociception.

Norepinephrine and serotonin involvement in nociceptive functions is supported by observations of analgesic effects of norepinephrine transporter (NET) and serotonin transporter (SERT) inhibitors such as amitriptyline. However, the relative contribution of NET and SERT to baseline nociception, as well as amitriptyline analgesia, is unclear. Amitriptyline and morphine analgesia in wild-type (WT) mice and littermates with gene knockout (KO) of SERT, NET or both transporters was conducted using the hotplate and tail-flick tests. Hypoalgesia was observed in NET KO mice, and to a lesser extent in SERT KO mice. The magnitude of this hypoalgesia in NET KO mice was so profound that it limited the assessment of drug-induced analgesia. Nonetheless, the necessary exclusion of these subjects because of profound baseline hypoalgesia strongly supports the role of norepinephrine and NET in basal nociceptive behavior while indicating a much smaller role for serotonin and SERT. To further clarify the role of NET and SERT in basal nociceptive sensitivity further experiments were conducted in SERT KO and NET KO mice across a range of temperatures. NET KO mice were again found to have pronounced thermal hypoalgesia compared to WT mice in both the hotplate and tail-flick tests, while only limited effects were observed in SERT KO mice. Furthermore, in the acetic acid writhing test of visceral nociception pronounced hypoalgesia was again found in NET KO mice, but no change in SERT KO mice. As some of these effects may have resulted from developmental consequences of NET KO, the effects of the selective NET blocker nisoxetine and the selective SERT blocker fluoxetine were also examined in WT mice: only nisoxetine produced analgesia in these mice. Collectively these data suggest that NET has a far greater role in determining baseline analgesia, and perhaps other analgesic effects, than SERT in mice.

Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis.

D-amphetamine (AMPH) down-regulates the norepinephrine transporter (NET), although the exact trafficking pathways altered and motifs involved are not known. Therefore, we examined the cellular and molecular mechanisms involved in AMPH-induced NET regulation in human placental trophoblast cells expressing the wild-type (WT)-hNET and the hNET double mutant (DM)-bearing protein kinase C (PKC)-resistant T258A + S259A motif. NET function and surface expression were significantly reduced in cells expressing WT-hNET but not in cells expressing hNET-DM following AMPH treatment. AMPH inhibited plasma membrane recycling of both WT-hNET and hNET-DM. In contrast, AMPH stimulated endocytosis of WT-hNET, and did not affect hNET-DM endocytosis. Although PKC or calcium/calmodulin- dependent kinase-II (CaMKII) inhibition or depletion of calcium failed to block AMPH-mediated down-regulation of WT-hNET, NET-specific blocker desipramine completely prevented AMPH-induced down-regulation. Furthermore, AMPH treatment had no effect on phospho-CaMKII immunoreactivity. The inhibitory potency of AMPH was highest on hNET-DM, intermediary on T258A and S259A single mutants and lowest on WT-hNET. Single mutants exhibited partial resistance to AMPH-mediated down-regulation. AMPH accumulation was similar in cells expressing WT-hNET or hNET-DM. The results demonstrate that reduced plasma membrane insertion and enhanced endocytosis account for AMPH-mediated NET down-regulation, and provide the first evidence that T258/S259 motif is involved only in AMPH-induced NET endocytosis that is desipramine-sensitive, but PKC and CaMKII independent.

Dysregulation of the norepinephrine transporter sustains cortical hypodopaminergia and schizophrenia-like behaviors in neuronal rictor null mice.

The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.

PKC links Gq-coupled receptors to DAT-mediated dopamine release.

The dopamine transporter (DAT) mediates dopamine uptake in many brain areas and has been suggested to underlie dopamine release in the substantia nigra pars reticulata. This release modulates basal ganglia output and motor performance. It is triggered by glutamatergic subthalamic afferents. We show here that activation of (G(q)-coupled) group I metabotropic glutamate receptors are sufficient to induce dopamine release in rat substantia nigra pars reticulata slices. Release was measured by amperometry and inhibited by a DAT antagonist. In a DAT-expressing cell line, dopamine release could be induced by activation of G(q)-coupled muscarinic receptors. Release was measured by HPLC and inhibited by a DAT antagonist. In both paradigms, activation of protein kinase C (PKC) was necessary and sufficient for dopamine release, suggesting that G(q)-coupled receptors can induce DAT-mediated dopamine release through PKC, and providing a physiological role for the known regulation of DAT-mediated release by PKC. Activation of muscarinic receptors and PKC also induced release from a cell line expressing the norepinephrine transporter, suggesting that transport-mediated release might be relevant in further brain areas.