Ubiquitous embryonic expression of the norepinephrine transporter.

We report that the norepinephrine transporter (NET) is expressed in avian and mouse embryos by numerous tissues that are derived from all three germ layers. In the nervous system, NET is expressed in the neuroepithelium of the brain and the spinal cord (ventral horn and floor plate), forming mesencephalic nuclei, neural crest, dorsal root ganglion, sympathetic ganglion and spinal nerve. Nonneuronal embryonic NET-expressing structures include the olfactory epithelium, the notochord, the somitic dermamyotome and mesenchymal cells in the limb bud. NET is expressed prominently in the cardiovascular system, including endothelial cells of forming blood vessels, the walls of the aorta and veins, the epicardium, myocardium and a subset of blood cells. The gut, lung buds, and in particular the kidneys, are intensely NET immunoreactive. Since neurotransmitters are known to affect proliferation, survival and differentiation of many mesenchymal cell types, NET function may be a physiologically relevant regulatory element in embryonic development. A working model is proposed for neurotransmitter transporter function in the embryo as a system for the concentration and targeted delivery of neurotransmitter.

Autocrine regulation of norepinephrine transporter expression.

The norepinephrine transporter (NET) is a neurotransmitter scavenger and site of drug action in noradrenergic neurons. The aim of this study was to identify mechanisms that regulate NET expression during the development of quail (q) sympathetic neuroblasts, which develop from neural crest stem cells. Neurotrophin-3 (NT-3) and transforming growth factor beta1 (TGF-beta1) cause an increase of qNET mRNA levels in neural crest cells. When combined, the growth factors are additive in increasing qNET mRNA levels. Both NT-3 and TGF-beta1 are synthesized by neural crest cells. Onset of NET expression precedes the onset of neural crest stem cell emigration from the neural tube. In older embryos, qNET is expressed by several crest-derived and noncrest tissues. The data show that qNET expression in presumptive sympathetic neurons is initiated early in embryonic development by growth factors that are produced by neural crest cells themselves. Moreover, the results support our previous observations that norepinephrine transport contributes to the regulation of the differentiation of neural crest stem cells into sympathetic neurons.

The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: a primordial carrier for catecholamines.

Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 microM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian catecholamine transporters. The combination of a DAT-like substrate selectivity and norepinephrine transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral catecholamine transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior.

Comparison of the pharmacological properties of cloned rat, human, and bovine norepinephrine transporters.

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [(3)H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

The human noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons.

Sequencing downstream of the C-terminal exon 14 of the human noradrenaline transporter (hNAT) gene reveals 5 consensus polyadenylation signals, several adenylate/uridylate-rich elements (AREs) and a new C-terminal exon, designated as exon 15. The tandemly arranged polyadenylation sites are in good conformity with the 3.6- and 5.8-kb hNAT mRNA transcripts. Expression of the alternatively spliced C-terminal exon 15 is shown by RT-PCR. This alternative splicing event proposes additional hNAT mRNA species of 2.4-3 kb in size. Corresponding NAT transcripts are found by Northern analysis of human SKN and rat PC12 cell RNA. Sequence comparison of the hNAT gene to two bovine NAT cDNAs shows the interspecies conservation of this alternative splicing event, the close relationship of human and bovine NAT genes, and implicates a functional role for the transporters C-terminal domain.

The rat norepinephrine transporter: molecular cloning from PC12 cells and functional expression.

The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [3H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters.

Systematic search for variation in the human norepinephrine transporter gene: identification of five naturally occurring missense mutations and study of association with major psychiatric disorders.

The complete coding region of the norepinephrine transporter (NET) gene was systematically screened for genetic variants in 137 unrelated individuals (including 46 probands with bipolar affective disorder and 45 schizophrenic probands, as well as 46 blood donors) using single-strand conformation analysis. We identified 13 DNA sequence variants, among them five missense substitutions. The missense substitutions Val69Ile, Thr99Ile, Val245Ile, Val449Ile, and Gly478Ser are located at putative transmembrane domains (TMD) 1, 2, 4, 9, and 10, respectively. The Thr99Ile substitution is at the 5th position of the putative leucine-zipper in TMD2. In a case-control study distribution of missense substitutions was found to be similar in 103 patients with bipolar affective disorder, in 228 schizophrenia patients and in 187 controls, indicating that presence of these variants is not causally related to major psychiatric diseases. The detection of a highly polymorphic silent 1287G/A polymorphism was utilized to demonstrate biallelic expression of the NET in adult human brain.

Molecular cloning and organization of the coding region of the human norepinephrine transporter gene.

A lambda phage genomic library was screened with the digoxygenin labeled cDNA of the human norepinephrine transporter (hNET). Six overlapping lambda clones were analysed by restriction enzyme analysis and sequencing of the exon-intron boundaries. The coding region of the hNET gene was found to be encoded by 14 exons, spanning 45 kb from the start to the stop codon, disrupted by 13 introns. The organization of the gene is highly homologous to other known neurotransmitter transporter genes. However, the hNET gene differs from the other genes in that it has an additional exon encoding the C-terminus of the protein. The gene structure shows two large introns in the 5'-region and a cluster of 11 exons in the 3'-region. All exon-intron junctions contain the gt/ag consensus splice site. Knowledge of the gene structure of the antidepressant-sensitive hNET should facilitate investigation of its potential role in psychiatric disorders.