Nisoxetine Binding Research Articles

Effects of SRI-32743, a Novel Quinazoline Structure-Based Compound, on HIV-1 Tat and Cocaine Interaction with Norepinephrine Transporter.

Prolonged exposure to HIV-1 transactivator of transcription (Tat) protein dysregulates monoamine transmission, a physiological change implicated as a key factor in promoting neurocognitive disorders among people living with HIV. We have demonstrated that in vivo expression of Tat in Tat transgenic mice decreases dopamine uptake through both dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex. Further, our novel allosteric inhibitor of monoamine transporters, SRI-32743, has been shown to attenuate Tat-inhibited dopamine transport through DAT and alleviates Tat-potentiated cognitive impairments. The current study reports the pharmacological profiles of SRI-32743 in basal and Tat-induced inhibition of human NET (hNET) function. SRI-32743 exhibited less affinity for hNET binding than desipramine, a classical NET inhibitor, but displayed similar potency for inhibiting hDAT and hNET activity. SRI-32743 concentration-dependently increased hNET affinity for [3H]DA uptake but preserved the Vmax of dopamine transport. SRI-32743 slowed the cocaine-mediated dissociation of [3H]Nisoxetine binding and reduced both [3H]DA and [3H]MPP+ efflux but did not affect d-amphetamine-mediated [3H]DA release through hNET. Finally, we determined that SRI-32743 attenuated a recombinant Tat1-86-induced decrease in [3H]DA uptake via hNET. Our findings demonstrated that SRI-32743 allosterically disrupts the recombinant Tat1-86-hNET interaction, suggesting a potential treatment for HIV-infected individuals with concurrent cocaine abuse.

Mutations of tyrosine 467 in the human norepinephrine transporter attenuate HIV-1 Tat-induced inhibition of dopamine transport while retaining physiological function.

Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [3H]dopamine and [3H]norepinephrine in PC12 cells expressing wild-type hNET and its mutants. Compared to wild-type hNET, neither of Y467H or Y467F altered Bmax and Kd values of [3H]WIN35,428 binding, whereas Y467H but not Y467F decreased the Bmax of [3H]nisoxetine binding without changes in Kd. Y467H also increased the affinity of nisoxetine for inhibiting [3H]dopamine uptake relative to wild-type hNET. Recombinant Tat1-86 (140 nM) induced a significant reduction of [3H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [3H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [3H]MPP+ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND.

Autoradiographic localization of [3H]-Nisoxetine binding sites in the CNS of male and female Japanese quail

BackgroundIn the central nervous system of mammals, transporters localized on the presynaptic nerve terminals regulate the reuptake of neurotransmitters. These transporters are selective for a specific neurotransmitter such as dopamine (DA) and norepinephrine (NE). Specifically in the synapse, the dopamine transporter (DAT) reuptakes DA and the norepinephrine transporter (NET) reuptakes NE. However previous research has found that avian species do not have a gene for DAT, and therefore, birds may be using the NET to clear both NE and DA from the synapse. The current study aimed to extend this finding by localizing NET expression in male and female Japanese quail (Coturnix japonica) brains using [3H]Nisoxetine, a selective NET blocker.ResultsHigh densities of binding sites were observed in the olfactory tubercle (OTu), the medial striatum (MSt), and the lateral striatum (LSt). Lower densities of binding sites were detected in the amygdala (AMY) and hypothalamus (Hyp), and low binding was found in the medial preoptic area (mPOA) and the pallium.ConclusionThe areas with the highest densities of NET are also areas that previous research has shown to have high levels of DA activity but low levels of NE innervation (e.g. striatum). The distribution of this reuptake transporter is consistent with the theory that NET acts to clear both DA and NE from the synapse.

3H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein.

Dysregulation of dopamine neurotransmission has been linked to the development of human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND). To investigate the mechanisms underlying this phenomenon, this study used an inducible HIV-1 transactivator of transcription (Tat) transgenic (iTat-tg) mouse model, which demonstrates brain-specific Tat expression induced by administration of doxycycline. We found that induction of Tat expression in the iTat-tg mice for either 7 or 14 days resulted in a decrease (∼30%) in the V max of [3H]dopamine uptake via both the dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex (PFC), which was comparable to the magnitude (∼35%) of the decrease in B max for [3H]WIN 35,428 and [3H]nisoxetine binding to DAT and NET, respectively. The decreased V max was not accompanied by a reduction of total or plasma membrane expression of DAT and NET. Consistent with the decreased V max for DAT and NET in the PFC, the current study also found an increase in the tissue content of DA and dihydroxyphenylacetic acid in the PFC of iTat-tg mice after 7 days' administration of doxycycline. Electrophysiological recordings in layer V pyramidal neurons of the prelimbic cortex from iTat-tg mice found a significant reduction in action potential firing, which was not sensitive to selective inhibitors for DAT and NET, respectively. These findings provide a molecular basis for using the iTat-tg mouse model in the studies of NeuroHIV. Determining the mechanistic basis underlying the interaction between Tat and DAT/NET may reveal novel therapeutic possibilities for preventing the increase in comorbid conditions as well as HAND. SIGNIFICANCE STATEMENT: Human immunodeficiency virus (HIV)-1 infection disrupts dopaminergic neurotransmission, leading to HIV-associated neurocognitive disorders (HANDs). Based on our in vitro and in vivo studies, dopamine uptake via both dopamine and norepinephrine transporters is decreased in the prefrontal cortex of HIV-1 Tat transgenic mice, which is consistent with the increased dopamine and dihydroxyphenylacetic acid contents in this brain region. Thus, these plasma membrane transporters are an important potential target for therapeutic intervention for patients with HAND.

Effects of abstinence from chronic cocaine self-administration on nonhuman primate dorsal and ventral noradrenergic bundle terminal field structures.

Repeated exposure to cocaine is known to dysregulate the norepinephrine system, and norepinephrine has also been implicated as having a role in abstinence and withdrawal. The goal of this study was to determine the effects of exposure to cocaine self-administration and subsequent abstinence on regulatory elements of the norepinephrine system in the nonhuman primate brain. Rhesus monkeys self-administered cocaine (0.3mg/kg/injection, 30 reinforcers/session) under a fixed-interval 3-min schedule of reinforcement for 100 sessions. Animals in the abstinence group then underwent a 30-day period during which no operant responding was conducted, followed by a final session of operant responding. Control animals underwent identical schedules of food reinforcement and abstinence. This duration of cocaine self-administration has been shown previously to increase levels of norepinephrine transporters (NET) in the ventral noradrenergic bundle terminal fields. In contrast, in the current study, abstinence from chronic cocaine self-administration resulted in elevated levels of [(3)H]nisoxetine binding to the NET primarily in dorsal noradrenergic bundle terminal field structures. As compared to food reinforcement, chronic cocaine self-administration resulted in decreased binding of [(3)H]RX821002 to α2-adrenoceptors primarily in limbic-related structures innervated by both dorsal and ventral bundles, as well as elevated binding in the striatum. However, following abstinence from responding for cocaine binding to α2-adrenoceptors was not different than in control animals. These data demonstrate the dynamic nature of the regulation of norepinephrine during cocaine use and abstinence, and provide further evidence that the norepinephrine system should not be overlooked in the search for effective pharmacotherapies for cocaine dependence.

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.

Angiotensin II stimulates sympathetic neurotransmission to adipose tissue

Angiotensin II (AngII) facilitates sympathetic neurotransmission by regulating norepinephrine (NE) synthesis, release, and uptake. These effects of AngII contribute to cardiovascular control. Previous studies in our laboratory demonstrated that chronic AngII infusion decreased body weight of rats. We hypothesized that AngII facilitates sympathetic neurotransmission to adipose tissue and may thereby decrease body weight. The effect of chronic AngII infusion on the NE uptake transporter and NE turnover was examined in metabolic (interscapular brown adipose tissue, ISBAT; epididymal fat, EF) and cardiovascular tissues (left ventricle, LV; kidney) of rats. To examine the uptake transporter saturation isotherms were performed using [3H]nisoxetine (NIS). At doses that lowered body weight, AngII significantly increased ISBAT [3H]NIS binding density. To quantify NE turnover, alpha-methyl-para-tyrosine (AMPT) was injected in saline-infused, AngII-infused, or saline-infused rats that were pair-fed to food intake of AngII-infused rats. AngII significantly increased the rate of NE decline in all tissues compared to saline. The rate of NE decline in EF was increased to a similar extent by AngII and by pair feeding. In rats administered AngII and propranolol, reductions in food and water intake and body weight were eliminated. These data support the hypothesis that AngII facilitates sympathetic neurotransmission to adipose tissue. Increased sympathetic neurotransmission to adipose tissue following AngII exposure is suggested to contribute to reductions in body weight.

Novel Structure–Function Information on Biogenic Amine Transporters Revealed by Site-Directed Mutagenesis and Alkylation

The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.

The Role of Cysteines and Histidins of the Norepinephrine Transporter

The thiol reagent N-ethylmaleimide (NEM) is known to inhibit irreversibly ligand binding by the norepinephrine transporter (NET), while the simultaneous presence of NET substrates or ligands protects from this inhibition. Therefore, cysteine residues located within the substrate binding pocket of the NET were assumed to play an important role in ligand binding. To examine which (if any) of the 10 cysteines (Cys) of the human (h) NET might be involved in transport and/or binding function, we mutated all hNET cysteines to alanine. Using transfected HEK293 cells we studied NEM effects on the hNET with respect to [(3)H]nisoxetine binding. Two cysteines (Cys176 and Cys185) within the extracellular loop of the NET have been proposed to form a disulfide bond. We could demonstrate that this is of crucial importance as corresponding hNET mutants, in which these cysteines have been replaced, showed a lack of plasma membrane expression. However, due to their oxidized state in the native NET protein, Cys176 and Cys185 may not be targets for NEM. All other Cys-to-Ala hNET mutants were fully active and showed no change in inhibition of [(3)H]nisoxetine binding by NEM. These observations clearly exclude cysteines as being involved in hNET ligand binding. Since NEM also interacts with histidin (His), we mutated all 13 histidins of the hNET to alanine and examined the NET mutants in functional and binding assays. His222 within the large extracellular loop of the transporter was identified as an interaction partner of NEM since in the corresponding hNET mutant NEM exhibited a significantly reduced inhibitory potency. Furthermore, we could show that histidins in position 296, 370 and 372 are important for nisoxetine binding, while His220, 441, 598 and 599 are crucial for plasma membrane expression of the hNET.

α2-Adrenergic agonists including xylazine and dexmedetomidine inhibit norepinephrine transporter function in SK-N-SH cells

α2-Adrenergic agonists simulate norepinephrine (NE) action on α2 receptors of sympathetic neurons to mediate feedback inhibition of NE release. These agents are used as valuable adjuncts for management of hypertension and for anesthesia. Their action, equivalent to NE on α2 adrenergic receptors, raises the question whether α2 agonists may also target NE transporters (NETs), another major control mechanism for noradrenergic neurotransmission. We thus investigated the effect of α2 agonists on transport of the NE analog, 131I-metaiodobenzylguanidine (MIBG). Results from this investigation showed that xylazine and dexmedetomidine dose-dependently blocked [3H]nisoxetine binding in neuron-like SK-N-SH cells. Furthermore, the agents acutely suppressed cellular MIBG uptake in a dose-dependent manner. This effect was uninfluenced by the α2 antagonist yohimbine, but was completely reversed by drug removal. There was no change in membrane NET density by the agents. Moreover, saturation analysis showed that xylazine and dexmedetomidine significantly increased Km without affecting Vmax, indicating competitive inhibition of MIBG transport. Thus, the α2 adrenergic agonists xylazine and dexmedetomidine, acutely suppress NET function through competitive inhibition of substrate transport, likely by direct interaction on a region that over-laps with the nisoxetine binding site.

Pentazocine Inhibits Norepinephrine Transporter Function by Reducing its Surface Expression in Bovine Adrenal Medullary Cells

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [3H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (−)-PTZ and (+)-PTZ inhibited [3H]NE uptake by adrenal medullary cells in a concentration-dependent (3 – 100 μM) manner. Eadie-Hofstee analysis of [3H]NE uptake showed that both PTZs caused a significant decrease in the Vmax with little change in the apparent Km, suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [3H]NE uptake induced by (−)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [3H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [3H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the Bmax without changing the apparent Kd. Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (−)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor–independent pathway.

Antidepressant-like drug effects in juvenile and adolescent mice in the tail suspension test: Relationship with hippocampal serotonin and norepinephrine transporter expression and function

Depression is a major health problem for which most patients are not effectively treated. This problem is further compounded in children and adolescents where only two antidepressants [both selective serotonin reuptake inhibitors (SSRIs)] are currently approved for clinical use. Mouse models provide tools to identify mechanisms that might account for poor treatment response to antidepressants. However, there are few studies in adolescent mice and none in juvenile mice. The tail suspension test (TST) is commonly used to assay for antidepressant-like effects of drugs in adult mice. Here we show that the TST can also be used to assay antidepressant-like effects of drugs in C57Bl/6 mice aged 21 (juvenile) and 28 (adolescent) days post-partum (P). We found that the magnitude of antidepressant-like response to the SSRI escitalopram was less in P21 mice than in P28 or adult mice. The smaller antidepressant response of juveniles was not related to either maximal binding (B max) or affinity (Kd) for [3H]citalopram binding to the serotonin transporter (SERT) in hippocampus, which did not vary significantly among ages. Magnitude of antidepressant-like response to the tricyclic desipramine was similar among ages, as were Bmax and K d values for [3H]nisoxetine binding to the norepinephrine transporter in hippocampus. Together, these findings suggest that juvenile mice are less responsive to the antidepressant-like effects of escitalopram than adults, but that this effect is not due to delayed maturation of SERT in hippocampus. Showing that the TST is a relevant behavioral assay of antidepressant-like activity in juvenile and adolescent mice sets the stage for future studies of the mechanisms underlying the antidepressant response in these young populations.

Differential adaptive changes on serotonin and noradrenaline transporters in a rat model of peripheral neuropathic pain

Serotonin and noradrenaline reuptake inhibitors have shown to produce antinociceptive effects in several animal models of neuropathic pain. In the present work, we have analyzed the density of brain and spinal serotonin and noradrenaline transporters (5-HTT and NAT) in a rat model of neuropathic pain, the spinal nerve ligation (SNL). Quantitative autoradiography revealed a significant decrease in the density of 5-HTT ([3H]citalopram binding) at the level of the lumbar spinal cord following 2 weeks of neuropathic surgery (lamina V, −40%: 6.01±0.64nCi/mg tissue in sham-animals vs 3.59±1.56 in SNL-animals; lamina X, −30%: 9.10±2.00 vs 6.40±1.93 and lamina IX, −22%: 12.01±2.41 vs 9.42±1.58). By contrast, NAT density ([3H]nisoxetine binding) was significantly increased (lamina I–II, +34%: 2.20±0.45 vs 2.96±0.65; lamina V, +57%: 1.34±0.28 vs 2.11±0.66; and lamina IX, +58%: 2.39±0.71 vs 3.78±1.10). At supraspinal structures, SNL induced adaptive changes only in the density of 5-HTT (septal nuclei, +33%: 10.18±2.03 vs 13.53±1.14; CA3 field of hippocampus, +18%: 6.94±1.01 vs 8.21±0.81; paraventricular thalamic nucleus, +21%: 15.18±1.88 vs 18.35±2.08; lateral hypothalamic area, +40%: 12.68±1.90 vs 17.8±2.55; ventromedial hypothalamic nuclei, +19%: 7.16±0.92 vs 8.55±0.40; and dorsal raphe nucleus, +15%: 35.22±3.88 vs 40.68±3.11). Thus, we demonstrate, in the SNL model of neuropathic pain, the existence of opposite changes in the spinal expression of 5-HTT (down-regulation) and NAT (up-regulation), and the presence of supraspinal adaptive changes (up-regulation) only on 5-HTT density. These findings may help understanding the pathogeny of neuropathic pain and the differential analgesic action of antidepressants targeting 5-HTT and/or NAT transporters.

Characterization of [3H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [3H]nisoxetine. However, traditional [3H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [3H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [3H]CFT binding to whole cells (at physiological [Na+] and 25°C) were similar to potencies observed in the [3H]NE uptake assay. Inhibition constants (Ki) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R2=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [3H]nisoxetine binding to membrane preparations (4°C for 3h, [Na+] at 295mM), the potency of nisoxetine and desipramine in inhibiting [3H]CFT binding became greater than that measured in a functional assay of [3H]NE uptake at physiological [Na+]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [3H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [3H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.

Endothelin-1 and -3 modulate the neuronal norepinephrine transporter through multiple signalling pathways in the rat posterior hypothalamus

We have previously reported that endothelin-1 and -3 modulate different steps of noradrenergic transmission in the hypothalamus. We showed that endothelins modify neuronal norepinephrine transport activity through the regulation of the kinetic constant and internalization. In the present work we sought to define the endothelin receptors and intracellular mechanisms involved in the down-regulation of neuronal norepinephrine uptake induced by endothelin-1 and -3 in the rat posterior hypothalamic region. Results showed that endothelin-1 reduced norepinephrine uptake through ET B receptors, whereas endothelin-3 through a non-conventional or atypical endothelin receptor. In both cases, the effect on norepinephrine uptake was coupled to protein kinase A and C as well as nitric oxide pathways. However, neither protein kinase G nor intracellular or extracellular calcium and calcium/calmodulin-dependent protein kinase II were involved. In addition, the same intracellular mechanisms participated in the reduction of nisoxetine binding (norepinephrine transporter internalization index) induced by both endothelins. Present findings reveal the underlying mechanisms involved in the regulation of the neuronal norepinephrine transporter by endothelins and further support the role of these peptides in the modulation of noradrenergic transmission at the presynaptic nerve endings in the posterior hypothalamus.

Stimulatory effects of the soy phytoestrogen genistein on noradrenaline transporter and serotonin transporter activity

We examined the effects of genistein, one of the major soy phytoestrogens, on the activity of noradrenaline transporter (NAT) and serotonin transporter. Treatment with genistein (10 nM-10 microM) for 20 min stimulated [(3)H]noradrenaline (NA) uptake by SK-N-SH cells. Genistein also stimulated [(3)H]NA uptake and [(3)H]serotonin uptake by NAT and serotonin transporter transiently transfected COS-7 cells, respectively. Kinetics analysis of the effect of genistein on NAT activity in NAT-transfected COS-7 cells revealed that genistein significantly increased the maximal velocity of NA transport with little or no change in the affinity. Scatchard analysis of [(3)H]nisoxetine binding to NAT-transfected COS-7 cells showed that genistein increased the maximal binding without altering the dissociation constant. Although genistein is also known to be an inhibitor of tyrosine kinases, daidzein, another soy phytoestrogen and an inactive genistein analogue against tyrosine kinases, had little effect on [(3)H]NA uptake by SK-N-SH cells. The stimulatory effects on NAT activity were observed by treatment of tyrphostin 25, an inhibitor of epidermal growth factor receptor tyrosine kinase, whereas orthovanadate, a protein tyrosine phosphatase inhibitor, suppressed [(3)H]NA uptake by NAT-transfected COS-7 cells. These findings suggest that genistein up-regulates the activity of neuronal monoamine transporters probably through processes involving protein tyrosine phosphorylation.

Assessment of the adrenergic effects of orphenadrine in rat vas deferens.

The peripheral adrenergic effects of orphenadrine, an antiparkinsonian drug, have been evaluated in the rat vas deferens to investigate whether these properties are the same as those of other phencyclidine ligands. In the low micromolar range, orphenadrine enhanced electrically-evoked and exogenous noradrenaline contractile responses in the epididymal portion of rat vas deferens. It also induced spontaneous activity that was inhibited by prazosin (1 microM) but not by atropine (20 nM). It inhibited accumulation of [3H]noradrenaline in rat vas deferens (IC50 = 14.2+/-2.3 microM). Orphenadrine competitively inhibited [3H]nisoxetine binding in rat vas deferens membranes (Ki = 1.05+/-0.20 microM). It can be concluded that orphenadrine, at low micromolar concentrations, interacts with the noradrenaline reuptake system inhibiting its functionality and thus potentiating the effect of noradrenaline.

Chronic oral nicotine increases brain [3H]epibatidine binding and responsiveness to antidepressant drugs, but not nicotine, in the mouse forced swim test

Smoking rates among depressed individuals is higher than among healthy subjects, and nicotine alleviates depressive symptoms. Nicotine increases serotonergic and noradrenergic neuronal activity and facilitates serotonin and noradrenaline release. In mice, acute nicotine administration enhances the activity of antidepressants in the mouse forced swim (mFST) and tail suspension tests. Here, we investigated if this action of nicotine is also reflected in a chronic treatment regimen. After chronic treatment with nicotine in the drinking water, mice were challenged with nicotine, duloxetine, citalopram, and reboxetine in the mFST. Additionally, 8-OH-DPAT- and clonidine-induced hypothermia was tested in vehicle- and nicotine-pretreated mice, as a measure of 5-HT(1A) and alpha(2)-adrenoceptor function, respectively. Finally, the effects on the brain expression levels of high- and low-affinity nicotinic acetylcholine receptors (nAChRs) and the transporters for serotonin (SERT) and noradrenaline (NET) were assessed using [(3)H]epibatidine, [(3)H]alpha-bungarotoxin, [(3)H]citalopram, and [(3)H]nisoxetine binding, respectively. In the mFST, nicotine-pretreated mice did not show altered response to the nicotine challenge, but increased responses to all three antidepressants tested were observed when compared to mice that had been administered drinking water without nicotine. There was no change in hypothermic responses to 8-OH-DPAT or clonidine. [(3)H]epibatidine binding was significantly increased in all brain regions investigated; whereas, [(3)H]alpha-bungarotoxin, [(3)H]citalopram, and [(3)H]nisoxetine binding were not altered, indicating that chronic oral nicotine increases the expression and/or affinity of high-affinity nAChRs, but not low-affinity nAChRs, SERT, or NET. It is suggested that the increased sensitivity to antidepressants after chronic nicotine exposure involves increased high-affinity nAChR-mediated neurotransmission.

Intracellular signaling mechanisms underlying the activity and internalization of the neuronal norepinephrine transporter by endothelin‐3 in the rat posterior hypothalamus

Brain norepinephrine (NE) is involved in relevant physiological functions regulated by the hypothalamus. We previously reported that endothelins (ET) participate in the short term regulation of NE uptake in the anterior and posterior hypothalamus by changing the kinetic and internalization of the neuronal NE transporter (NET). In the present work we studied the mechanisms involved in the regulation of NET by ET‐3 in the rat posterior hypothalamic region (PHR). Experiments were performed in vitro and neuronal NE uptake and nisoxetine binding were used as indexes of activity and internalization of NET, respectively. Results showed that ET‐3 reduced neuronal NE uptake through the activation of a non conventional ET receptor involving PKC, PKA and PKG pathways. Furthermore ET‐3 enhanced NET internalization. However incubation with inhibitors of PKC (GF‐109203x), PKA (H‐89) or PKG (KT5823) inhibited ET‐3 response on NET internalization. Present findings suggest that the effect of ET‐3 on NET in the rat PHR is mediated by a non‐conventional ET receptor coupled to various intracellular pathways including PKC, PKA and PKG.(UBACyT B079, CONICET‐PIP 5929, ANPCyT PICT‐38333)

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.