Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transportere

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of IIE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in V-max values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

18F-Labeled Presynaptic Dopaminergic Tracers. Synthesis, Radiochemical Analyses, and Preclinical Evaluation of [18F]FDOPA and [18F]CFT

Dysfunction of the dopaminergic system in brain is involved in several pathological conditions such as Parkinson’s disease and depression. 2β-Carbomethoxy-3β-(4-[18F] fluorophenyl)tropane ([18F]CFT) and 6-[18F]fluoro-L-dopa ([18F]FDOPA) are tracers for imaging the dopaminergic function with positron emission tomography (PET). Peripheral uptake of [18F]FDOPA is also used in the localization and diagnosis of neuroendocrine tumors. [18F]FDOPA and [18F]CFT can be synthesized by electrophilic fluorodestannylation. However, the specific radioactivity (SA) in the electrophilic fluorination is low with traditional synthetic methods. In this study, [18F]FDOPA and [18F]CFT were synthesized using post-target-produced [18F]F2 as an electrophilic fluorination agent. With this method, tracers are produced with sufficient SA for neuroreceptor studies. Specific aims in this study were to replace Freon-11 in the production of [18F]FDOPA due to the ozone depleting properties of this solvent, to determine pharmacological specificity and selectivity of [18F]CFT with respect to monoamine transporters, and to compare the ability of these tracers to reflect the degree of nigral neuronal loss in rats in which the dopaminergic system in the brain had been unilaterally destroyed by 6- OHDA. Post-target-produced [18F]F2 was successfully used in the production of [18F]FDOPA and [18F]CFT. The SA achieved was substantially higher than in previous synthetic methods. Deuterated compounds, CD2Cl2, CDCl3 and C3D6O, were found to be suitable solvents for replacing Freon-11. Both [18F]FDOPA and [18F]CFT demonstrated nigrostriatal dopaminergic hypofunction and correlated with the number of nigral dopaminergic neurons in the 6-OHDA lesioned rat. However, the dopamine transporter (DAT) tracer [18F]CFT was more sensitive than the dopamine synthesis tracer [18F]FDOPA in detecting these defects because of the higher non-specific uptake of [18F]FDOPA. [18F]CFT can also be used for imaging the norepinephrine transporter (NET) because of the specific uptake into the locus coeruleus. The observation that [18F]CFT exhibits specific uptake in the pancreas warrants further studies in humans with respect to potential utility in pancreatic imaging

Investigação do envolvimento de subtipos de adrenoceptores α1 no efeito anti-imobilidade da imipramina no teste de suspensão pela cauda

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Bupropion treatment increases epididymal contractility and impairs sperm quality with no effects on the epididymal sperm transit time of male rats

Bupropion is a dopamine (DA) and norepinephrine (NE) reuptake inhibitor used as smoking cessation and antidepressant drug with a lower incidence of male sexual dysfunction. We showed previously that sibutramine, a norepinephrine/serotonine reuptake inhibitor, reduced male rat fertility. As there are no studies evaluating the impact of bupropion treatment on spermatic parameters and male fertility, we evaluated the effects of bupropion treatment (15 and 30 mg kg(-1), 30 days) on sexual behavior, spermatic parameters and fertility of male Wistar rats and on the epididymal duct in vitro contractility. Bupropion 15 mg kg(-1) increased the serum luteinizing hormone level and the epididymal duct contractility, but the sperm quality was not affected. At 30 mg kg(-1) bupropion impaired sperm quality increasing the incidence of non-progressive sperm. The male sexual behavior and fertility were not modified at both bupropion doses. These results, in rats, suggest the importance of studies evaluating the effects of bupropion on the human male sperm quality.

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters

Cone snails have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in normal and diseased states. A number of these peptides have shown efficacy in vivo, including inhibitors of calcium channels, the norepinephrine transporter, nicotinic acetylcholine receptors, NMDA receptors and neurotensin receptors, with several having undergone pre-clinical or clinical development for the treatment of pain.

Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Molecular mechanisms of cocaine reward: Combined dopamine and serotonin transporter knockouts eliminate cocaine place preference

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Influence Of The Major Nitrite Transporter Nirc On The Virulence Of A Swollen Head Syndrome Avian Pathogenic E. Coli (apec) Strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.

Contralateral cortical response of a subpopulation of interneurons and the glial glutamate transporter GLT1 to an ischemic core

Introduction: Cerebral ischemia is an important cause of brain lesion in humans. The target in research has been the ischemic core or the penumbra zones; little attention has been given to areas outside the core or the penumbra but connected with the primary site of injury. Objective: Evaluate the laminar response of a subpopulation of gabaergic cells, those that are parvalbumin (PV) positive and the astrocytes through the expression of the glial transporter GLT1 on the contralateral cortex to an ischemic core. Methodology: For this purpose we used the medial cerebral artery occlusion model in rats. The artery was occluded for 90 minutes and the animals were sacrificed at 24 and 72 hours post-ischemia. The brains were removed, cut in a vibratome at 50 microns and incubated with the primary antibodies against PV or GLT1. Sections were developed using the vectastain Kit. In control tissue the primary antibody was omitted. Results: When compared with control animals, treated ones show a decrease in the expression of GLT1, especially in layers III and IV of the contralateral cortex to the ischemic core. PV positive cells increases in layers II and V. Conclusion: Increases in the expression of PV cells could correspond to an adaptation associated with glutamate increases in the synaptic compartment. These increases may be due to decreases in the expression of GLT1 transporter, that could not remove the glutamate present in the synaptic cleft, generating hyperactivity in the contralateral cortex. These changes could represent an example of neuronal and glial plasticity in remote areas to an ischemic core but connected to the primary site of injury.

The Monocarboxylate Transporter 8 and L-Type Amino Acid Transporters 1 and 2 Are Expressed in Mouse Skeletons and in Osteoblastic MC3T3-E1 Cells

Background: Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. Methods: The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. Results: MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. Conclusions: This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

The serotonin transporter gene and Parkinson's disease

Dysfunction in the serotonin (5-hydroxytryptamine) system and reduced serotonin concentrations have been reported in patients with Parkinson's disease (PD). Serotonin concentrations in neural tissue are controlled by a presynaptic serotonin transporter protein that is encoded by a single gene. Therefore, we investigated whether a polymorphic region in the serotonin transporter gene is associated with PD. Three variable-number tandem repeat (VNTR) elements of the serotonin transporter gene were detected by polymerase chain reaction, those with 9, 10, 11 and 12 copies of the repeat element. The 10-copy VNTR element was significantly less common in patients with PD than controls in the univariate analysis (p < 0.05). Logistic regression analysis revealed no significant differences between patients (n = 198) and controls (n = 200) in the distribution frequencies of 9-and 12-copy alleles and combined genotypes (odds ratio = 1.20; p = 1.71). A positive family history of PD was a strong predictor of disease risk (odds ratio = 2.98; 95% confidence interval 1.51-5.87; p = 0.001). Although slight differences were observed between patient and control groups, these data suggest that defects in serotonin concentrations in patients with PD are unlikely to be due to polymorphisms in the serotonin transporter gene in this large Australian cohort; however, the inverse association observed with the 10-copy allele warrants further investigation. Copyright (C) 2000 S. Karger AG, Basel.