Chromosomal localization of the human D3 dopamine receptor gene

Summary A novel dopamine D₃ receptor gene that may be involved in psychiatric diseases has recently been characterized. It has been assigned to chromosome 3 by hybridization with a D₃ receptor probe to human sorted chromosomes, and localized to band 3q 13.3 by in situ hybridization.     

The distribution of a dopamine D2 receptor mRNA in rat brain

Based on the recently reported sequence of a dopamine D2 receptor cloned from rat brain, we prepared a series of cDNA probes to determine the distribution of mRNA encoding this receptor. Within the forebrain, D2 receptor mRNA is abundant in the caudate-putamen, accumbens nucleus and olfactory tubercle. Moderate to low levels of mRNA are observed in the medial habenular nucleus, diagonal band, lateral septal nucleus, claustrum, dorsal endopiriform nucleus, and entorhinal cortex. In the mesencephalon, D2 receptor mRNA is abundant within the substantia nigra, pars compacta, and the ventral tegmental area. Comparison of the distribution of the mRNA and ligand binding indicates that both presynaptic and postsynaptic D2 receptors of the nigrostriatal, mesolimbic and mesocortical pathways are derived from the same mRNA.     

Chromosomal localization of three human D5 dopamine receptor genes

It is currently thought that genetic predisposition to imbalances in dopaminergic transmission may underlie several neurological disorders, including schizophrenia, manic depression, Tourette syndrome, Parkinson disease, Huntington disease, and alcohol abuse. Originally two receptors, D1 and D2, were thought to account for all of the pharmacological actions of dopamine. However, through homology screening three additional genes, D3, D4, and D5, and two pseudogenes closely related to D5 have been characterized. To begin our genomic and evolutionary analyses of the human D5 dopamine receptor gene and its two pseudogenes, we have mapped each of them to their respective chromosomes. By combining in situ hybridization results with sequence analysis of PCR products from microdissected chromosomes, somatic cell hybrids, and radiation hybrids, we have assigned DRD5 (the locus containing the functional human D5 receptor gene) to chromosome 4p16.1, DRD5P1 (the locus containing D5 pseudogene 1) to chromosome 2p11.1-p11.2, and DRD5P2 (the locus of D5 pseudogene 2) to chromosome 1q21.1.     

Orexin-A up-regulates dopamine D2 receptor and mRNA in the nucleus accumbens Shell

Molecular Biology Reports - Orexins-A (OrxA) and -B (OrxB) neuropeptides are synthesized by a group of neurons located in the lateral hypothalamus and adjacent perifornical area, which send their...     

Immunostimulating effect of D2 dopamine receptor agonist quinpirole

Immunostimulating activity of specific postsynaptic D2 receptor agonist quinpirole (Ly 171555) was studied in CBA mice. The drug was administered intraperitoneally in a dose of 1 mg/kg 30 min before the immunization with SRBC (5 x 10(8)). Considerable stimulation of rosette- and plaque-forming cell number was observed at the early stage (3-d day) and at more late stage of the immune response (5-th day). The increase of rosette- and plaque-forming cell number was provided by IgM-reaction enhancement.     

Seasonal relationships between dopamine D1 and D2 receptor and equine FSH receptor mRNA in equine ovarian epithelium

Dopamine (DA) blockade during anestrus or early spring transition can facilitate ovarian recrudescence and advance the timing of the first ovulation of the season. Some laboratories have reported variable results using DA antagonists to stimulate follicular growth during the mid-portion of the anestrual period. Differences in DA antagonist efficacy may be due to the FSH secretory status of the anestrous mare and the presence or absence of functional ovarian FSH receptors. We hypothesize that direct ovarian dopaminergic input can affect follicular growth through regulation of FSH receptor (FSHr) populations. To investigate this, the amount of DA D1 and D2 receptor (D1r, D2r) and FSHr mRNA was quantified in ovarian tissues in anestrous and mares expressing estrus at typical intervals that are detected during the breeding season. Ovaries (n=26) were collected from 10 anestrous mares and 13 mares that had initiated estrous cycles (n=8 luteal; n=5 follicular phase). The quantity of D1r and D2r mRNA and FSHr mRNA was determined in cortex of both groups and granulosa/theca (those having initiated estrous cycles) tissues by semi-quantitative polymerase chain reaction using the comparative cycle time method. The reference gene was glyceraldehyde-3-phosphate dehydrogenase. The fold-change for each sample was calculated based on a calibrator sample. Fold-change values for D1r and D2r were the dependent variable and tissue was the independent variable in a one-way ANOVA. Results of fold-change in FSHr were compared by ANCOVA due to unequal sample sizes from each mare. Correlations between receptors within each tissue type were determined. For each receptor type and tissue, correlations between follicular and luteal phases were determined. The fold-change of D1r mRNA was less than D2r mRNA in all tissue types and between seasons. The quantity of D2r message in ovarian cortex was greater (p<0.05) during anestrus than after estrous cycles had been initiated. Fold-change in D2r in granulosa/theca was not different dependant on estrous cycle phase or follicle size. Quantity of FSHr mRNA was less in anestrous ovarian cortex and greater after estrous cycles had been initiated. FSHr mRNA fold-change in the ovarian cortex after estrous cycle initiation was not different between estrous cycle phases, but was greater in smaller (<30mm) follicles compared with larger (>/=30mm) follicles. We have demonstrated an inverse temporal relation between ovarian D2r and FSHr in mares dependant upon season. The functional significance of this relationship deserves further study.     

Structural insights into the human D1 and D2 dopamine receptor signaling complexes

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Voltage-sensitivity at the human dopamine D2S receptor is agonist-specific

Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled receptors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evidence that voltage-sensitivity at the dopamine D_2S receptor is agonist-specific. Whereas the potency of dopamine at the D_2S receptor is decreased by depolarization, the potencies of β-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein subtypes or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta

Summary The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na⁺, K⁺-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg²⁺-ATPase).Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor.It is concluded that the strontium capture technique gave a reliable localization for Na⁺, K⁺-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

The effect of dopamine D1 and D2 receptor agonists on inositol phosphate turnover in rat striatal slices

There is a lot of controversy in the literature about the role of dopamine D1 and D2 receptor stimulation on the turnover of phosphoinositols, and phosphoinositols are one of the important second messenger. In order to resolve this controversy, the effect of dopamine receptor stimulation on turnover of phosphoinositols was studied by estimation of the accumulation of individual labelled inositols in rat striatal slices which were prelabelled with [3H]myoinositol. Incubation of the prelabelled striatal slices with 1 microM of quinpirole, D2 specific agonist or with 1 microM of SKF-38393, D1 specific agonist, did not affect the accumulation of basal level of either inositol monophosphate, or inositol biphosphate, or inositol triphosphate. In addition, in conclusion of D1 specific antagonist cis-flupentixol or D2 specific antagonist sulpiride did not affect the basal levels of inositol phosphates. The activity of enzyme phospholipase-C which produces these inositol phosphates was also measured in rat striatal membrane. Incubation of rat striatal membrane with either agonist quinpirole or SKF-38393 did not change the basal level of phospholipase C. Our data thus indicate that occupation of dopamine receptors did not affect the inositol phosphate system in rat striatum.     

Structure and expression of human and rat D2 dopamine receptor genes.

D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.     

Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor

Dear Editor, Dopamine acts as an essential neurotransmitter whose signal-ing is conducted through five G protein-coupled receptors(GPCRs),dopamine D1 to D5 rece...     

Ca(2+)-activated ATPase of the mouse chorioallantoic placenta: developmental expression, characterization and cytohistochemical localization

A membrane-associated, Ca(2+)-activated, Mg(2+)-dependent ATPase activity has been identified in the mouse chorioallantoic placenta. The enzyme activity is expressed and increases as a function of gestation. Biochemical characterization shows that the enzyme is highly specific for Ca2+ and nucleotide triphosphates, with a Km of 0.97 mM [Ca2+] and a Vmax of 1.05 nmol Pi released mg-1 placental protein min-1. The mouse placental Ca(2+)-ATPase activity has a pI of approximately 6.8, and corresponds to two apparent Mr values of 118 and 150 x 10(3), based on Ferguson analysis of non-denaturing electrophoretograms. Enzyme activity is inhibited by phenothiazin (suggesting a calmodulin dependence), vanadate, erythrosin B and quercetin, but not by ouabain or levamisole. Enzyme cytohistochemistry revealed that the Ca(2+)-ATPase is localized to polyploid trophoblastic cells of the mouse inner placenta. These results suggest that the enzyme may be a functional component of transplacental calcium transport during mouse embryonic development.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization

 In order to improve the identification and characterization of dopaminoceptive neurons, the rat brain was mapped for D₁ dopamine receptor mRNA by non-radioactive in situ hybridization (ISH) with a 45mer digoxigenin-labeled oligonucleotide probe. The specificity of the results was controlled with the help of a 396-bp D₁ receptor riboprobe. Labeled hybrids were visualized with an alkaline phosphatase-coupled anti-digoxigenin antibody. The high resolution obtained permitted individual labeled cells to be identified and to distinction between cell bodies and processes. D₁ mRNA was largely confined to neurons. With the exception of ependymal cells, glial cells were not distinctly labeled. Subcellularly, D₁ mRNA was localized to perikarya but not to dendrites or axons. D₁ receptor-expressing neurons were present in all of the known terminal fields of mesencephalic or diencephalic dopaminergic neurons. However, D₁ message was also detected in brain areas which are not known to contain D₁ ligand binding sites or in which the presence or the cellular source of this receptor subtype had previously not been unequivocally established, such as the hippocampus or cerebellar cortex. Moreover, labeled neurons were present in regions not known to receive dopaminergic projections, such as the thalamic and some brainstem nuclei. We conclude that this ISH technique provides a considerable gain in sensitivity and resolution with regard to neurotransmitter receptor mapping. Accepted: 25 August 1997     

The dopamine D2 receptor gene in lamprey, its expression in the striatum and cellular effects of D2 receptor activation

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates.     

Dopamine transporter cell surface localization facilitated by a direct interaction with the dopamine D2 receptor

Altered synaptic dopamine levels have been implicated in several neurological/neuropsychiatric disorders, including drug addiction and schizophrenia. However, it is unclear what precipitates these changes in synaptic dopamine levels. One of the key presynaptic components involved in regulating dopaminergic tone is the dopamine transporter (DAT). Here, we report that the DAT is also regulated by the dopamine D2 receptor through a direct protein-protein interaction involving the DAT amino-terminus and the third intracellular loop of the D2 receptor. This physical coupling facilitates the recruitment of intracellular DAT to the plasma membrane and leads to enhanced dopamine reuptake. Moreover, mice injected with peptides that disrupt D2-DAT interaction exhibit decreased synaptosomal dopamine uptake and significantly increased locomotor activity, reminiscent of DAT knockout mice. Our data highlight a novel mechanism through which neurotransmitter receptors can functionally modulate neurotransmitter transporters, an interaction that can affect the synaptic neurotransmitter levels in the brain.