Further evidence for the involvement of D2, but not D1 dopamine receptors in dopaminergic control of striatal cholinergic transmission

The relative involvement of D₁ (cyclase linked) and D₂ dopamine receptors in dopaminergic control of striatal cholinergic transmission has been investigated in the rat by comparing the effects of SKF 38393 and LY 141865 (which act as specific agonists at D₁ and D₂ dopamine receptors, respectively) on striatal acetylcholine and dopamine metabolite concentrations and on the potassium-evoked release of ³H-acetylcholine from rat striatal slices. LY 141865 given systemically produced a dose-dependent increase in acetylcholine concentrations and a concomitant reduction of homovanillic and dihydroxyphenylacetic acid levels in the striatum (ED₅₀ 0.1 mg/kg) whereas SKF 38393 (1–30 mg/kg) did not. SKF 38393 (30 mg/kg) also failed to modify the LY 141865 (1 mg/kg) induced alterations of striatal acetylcholine and dopamine metabolite levels when given concomitantly with the latter compound. In experiments $\text{in vitro}$, LY 141865 reduced (EC₅₀ 0.14 μM), whereas SKF 38393 (up to 100 μM) failed to affect, the potassium-evoked release of ³H-acetylcholine from striatal slices. When given concomitantly with LY 141865, SKF 38393 (10 μM) did not modify the ability of the former compound to diminish striatal ³H-acetylcholine release. Finally, SKF 38393 also failed to affect the release of striatal ³H-acetylcholine after chemical lesion of the nigro-striatal dopaminergic pathway. The present results provide evidence for the involvement of D₂ but not D₁ dopamine receptors in dopaminergic control of striatal cholinergic transmission and indicate that D₁ dopamine receptors do not exert any modulatory influence on D₂ dopamine receptor mediated dopaminergic transmission.     

Coupling of D1 and D5 dopamine receptors to multiple G proteins

Dopamine receptors are a subclass of the super family of G protein-coupled receptors, that transduce their effects by coupling to specific G proteins. Within the dopamine receptor family, the adenylyl cyclase stimulatory receptors include the D₁ and D₅ subtypes. The D₁ and D₅ dopamine receptors are genetically distinct, sharing >80% sequence homology within the highly conserved seven transmembrane spanning domains, but displaying only 50% overall homology at the amino acid level. When expressed in transfected GH₄C₁ rat pituitary cells, both D₁ and D₅ receptors stimulate adenylyl cyclase and have identical affinities toward dopaminergic agonists and antagonists. In order to analyze specific signaling pathways mediated by activation of either D₁ or D₅ receptors, we have identified the G proteins that are coupled to these receptors. Through functional analyses and competition binding studies, and from immunoprecipitation techniques, using antisera against the various α subunits of G proteins, we have established that both D₁ and D₅ receptors couple to G_sα. In addition, D₁ receptors are also coupled to G_oα. Since G_oα has been implicated in the regulation of Ca²⁺, K⁺, and Na⁺ channels, this finding would suggest that D₁ receptors can mediate the functional activity of these ion channels. There is also evidence to indicate that D₅ receptors couple to G_zα, a novel G protein abundantly expressed in neurons. Thus, despite similar pharmacological properties, such differential coupling of D₁ and D₅ receptors to G proteins other than G_sα, indicates that dopamine can transduce varied signaling responses upon the simultaneous stimulation of both these receptors.     

Enhanced Dopamine D1 and D2 Receptor Gene Expression in the Hippocampus of Hypoglycaemic and Diabetic Rats

Hypoglycaemic coma and brain injury are potential complications of insulin therapy. Hippocampal neurons are particularly vulnerable to hypoglycaemic stress leading to memory impairment. In the present article, we have investigated the dopamine (DA) content, homovanillic acid (HVA)/DA turnover ratio, DA D₁ and DA D₂ receptors in the hippocampus of insulin-induced hypoglycaemic (IIH) and streptozotocin induced diabetic rats where brain functions are impaired. The DA content decreased significantly in hippocampus of diabetic, diabetic +IIH and control +IIH rats compared to control. The HVA/DA turnover ratio also increased significantly in diabetic, diabetic +IIH and control +IIH rats compared to control. Scatchard analysis using [³H] DA in the hippocampus showed a significant increase in DA receptors of diabetic, diabetic +IIH and control +IIH rats with decreased affinity. Gene expression studies using Real-time PCR showed an increased expression of DA D₁ and DA D₂ receptors in the hippocampus of hypoglycaemic and diabetic rats. Our results indicate that the dopaminergic system is impaired in the hippocampus of hypoglycaemic and hyperglycaemic rats impairing DA related functions of hippocampus. We observed a prominent dopaminergic functional disturbance in the hypoglycaemic condition than in hyperglycaemia compared to control. This dopaminergic dysfunction in hippocampus during hypoglycaemia and hyperglycaemia is suggested to contribute to cognitive and memory deficits. This will have clinical significance in the treatment of diabetes.     

Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D1 receptors

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/β, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D₁ receptor. Here, we report on the cloning of two further dopamine D₁ receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D₁-like receptors but shows some pharmacological similarities to invertebrate “DOP1” dopamine D₁-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate “INDR”-like dopamine D₁-like receptors.     

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

Asynchrony in the Diurnal Rhythms of Dopaminergic D2 Receptors in Different Brain Regions of the Rat

Diurnal variations of dopaminergic D₂ receptors have been described in the striatum of rats, while other dopaminergic regions remain unstudied. Diurnal variations of dopamine D₂ receptors in the striatum, frontal cortex, and amygdala of the rat, were characterized by the stereospecific binding of [³H]-spiperone. Clear rhythms were found in all these areas, but asynchronous to each other. Striatal receptors had diurnal variations with a single peak at 00:00 hours. Frontal cortex receptors showed two peaks at 00:00 and 12:00 hours. Amygdaline complex receptors had two peaks at 18:00 and 06:00 hours. Saturation binding curves and their Scatchard analysis indicated that the diurnal variations in [³H]-spiperone binding are related to changes in receptor density rather than its affinity. The diurnal variations asynchrony in [³H]-spiperone binding to dopaminergic D₂ receptors from different neural regions, suggest different regulation in each area. Other functional implications of these rhythms remains to be established.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

Distribution of Dopamine,its Metabolites,and D1 and D2 Receptors in Heterozygous And Homozygous Weaver Mutant Mice

In weaver mice, besides a postnatal cerebellar developmental anomaly probably caused by alterations of an inwardly rectifying K⁺ channel, there is a progressive loss of mesencephalic dopaminergic neurons. To further evaluate this deficit, endogenous dopamine and its metabolites were measured in 22 brain regions from heterozygous (wv/+) and homozygous (wv/wv) mutants, and compared to wild type (+/+) mice. In both wv/+ and wv/wv mutants there were profound dopamine depletions in all regions; these changes were accompanied by decreases in metabolites but with an increase of turnover indexes. Dopamine D₁ and D₂ receptors were examined by autoradiography, and their distribution was conserved. The results show that the dopaminergic deficit is widespread to all areas of innervation, and is probably compensated for by an increased turnover. Abnormal developmental growth signals, or aberrant cellular responses, may result in defective neurite formation of the midbrain dopaminergic neurons, leading to their postnatal death.     

Pharmacological Characterization of Mammalian D1 and D2 Dopamine Receptors Expressed in Drosophila Schneider‐2 Cells

Abstract

Mammalian D₁ and D₂ dopamine receptors were stably expressed in Drosophila Schneider‐2 (S2) cells and screened for their pharmacological properties. Saturable, dose‐dependent, high affinity binding of the D₁‐selective antagonist [³H]SCH‐23390 was detected only in membranes from S2 cells induced to express rat dopamine D₁ receptors, while saturable, dose‐dependent, high affinity binding of the D₂‐selective antagonist [³H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D₂ receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D₁ and D₂ receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D₁ receptor bound the (+)‐enantiomer of the D₁‐selective agonist SKF38393 with higher affinity than the (?)‐enantiomer, while the dopamine D₂ receptor bound the (?)‐enantiomer of the D₂‐selective agonist norpropylapomorphine with higher affinity than the (+)‐enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPγS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D₁ and D₂ receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D₁ and D₂ dopamine receptors free of promiscuous G protein contaminants.     

D1-Like Dopamine Receptor-Mediated Function in Congenic Mutants with D1 vs. D5 Receptor “Knockout”

Current understanding of the functional roles of individual dopamine D₁-like [D₁, D₅] and D₂-like [D_2L/S, D₃, D₄] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D₁ and D₅ receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion (“knockout”) of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D₁ mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D₁ mutants on a mixed genetic background; D₁ knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D₁ receptor appears to regulate specific topographies of orofacial movement in the mouse as these are “sculpted” in a time-dependent manner. Although the well-recognized role of the D₁-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D₁ receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D₅ counterparts as evidenced in the phenotype of congenic D₅ mutants.     

Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D₁-like family of dopamine receptors and inputs of aversion coming from neurons containing the D₂-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D₁ reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D₂ receptors function, we present evidence of σ₁ receptor molecular and functional interaction with dopamine D₂ receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D₂ receptors (the long isoform of the D₂ receptor) can complex with σ₁ receptors, a result that is specific to D₂ receptors, as D₃ and D₄ receptors did not form heteromers. We demonstrate that the σ₁-D₂ receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ₁ -D₂ receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ₁ knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D₂ receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D₁ and D₂ receptor containing neurons in the brain.     

Dopamine D1, D2, D3 Receptors,Vesicular Monoamine Transporter Type-2 (VMAT2) and Dopamine Transporter (DAT) Densities in Aged Human Brain

The dopamine D₁, D₂, D₃ receptors, vesicular monoamine transporter type-2 (VMAT2), and dopamine transporter (DAT) densities were measured in 11 aged human brains (aged 77–107.8, mean: 91 years) by quantitative autoradiography. The density of D₁ receptors, VMAT2, and DAT was measured using [³H]{"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, [³H]dihydrotetrabenazine, and [³H]WIN35428, respectively. The density of D₂ and D₃ receptors was calculated using the D₃-preferring radioligand, [³H]WC-10 and the D₂-preferring radioligand [³H]raclopride using a mathematical model developed previously by our group. Dopamine D₁, D₂, and D₃ receptors are extensively distributed throughout striatum; the highest density of D₃ receptors occurred in the nucleus accumbens (NAc). The density of the DAT is 10–20-fold lower than that of VMAT2 in striatal regions. Dopamine D₃ receptor density exceeded D₂ receptor densities in extrastriatal regions, and thalamus contained a high level of D₃ receptors with negligible D₂ receptors. The density of dopamine D₁ linearly correlated with D₃ receptor density in the thalamus. The density of the DAT was negligible in the extrastriatal regions whereas the VMAT2 was expressed in moderate density. D₃ receptor and VMAT2 densities were in similar level between the aged human and aged rhesus brain samples, whereas aged human brain samples had lower range of densities of D₁ and D₂ receptors and DAT compared with the aged rhesus monkey brain. The differential density of D₃ and D₂ receptors in human brain will be useful in the interpretation of PET imaging studies in human subjects with existing radiotracers, and assist in the validation of newer PET radiotracers having a higher selectivity for dopamine D₂ or D₃ receptors.     

1-substituted apomorphines as potent dopamine agonists

A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D₁, D_2L or D₃ receptor subtypes. All studied compounds had affinities in nanomolar range for D_2L and D₃ receptors and the change of the nature of substituent in position 1 had only moderate effect. D₁ receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.     

D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

Dopamine receptor localization in the mammalian retina

After a short history of dopamine receptor discovery in the retina and a survey on dopamine receptor types and subtypes, the distribution of dopamine receptors in the retinal cells is described and correlated with their possible role in cell and retinal physiology. All the retinal cells probably bear dopamine receptors. For example, the recently discovered D_1B receptor has a possible role in modulating phagocytosis by the pigment epithelium and a D₄ receptor is likely to be involved in the inhibition of melatonin synthesis in photoreceptors. Dopamine uncouples horizontal and amacrine cell-gap junctions through D₁-like receptors. Dopamine modulates the release of other transmitters by subpopulations of amacrine cells, including that of dopamine through a D₂ autoreceptor. Ganglion cells express dopamine receptors, the role of which is still uncertain. Müller cells also are affected by dopamine. A puzzling action of dopamine is observed in the ciliary retina, in which D₁- and D₂-like receptors are likely to be involved in the cyclic regulation of intraocular pressure. Most of the dopaminergic actions appears to be extrasynaptic and the signaling pathways remain uncertain. Further studies are needed to better understand the multiple actions of dopamine in the retina, especially those that implicate rhythmic regulations.     

Effects of Dopamine D2 Receptor Partial Agonist Antipsychotic Aripiprazole on Dopamine Synthesis in Human Brain Measured by PET with L-[β-11C]DOPA

Dopamine D₂ receptor partial agonist antipsychotic drugs can modulate dopaminergic neurotransmission as functional agonists or functional antagonists. The effects of antipsychotics on presynaptic dopaminergic functions, such as dopamine synthesis capacity, might also be related to their therapeutic efficacy. Positron emission tomography (PET) was used to examine the effects of the partial agonist antipsychotic drug aripiprazole on presynaptic dopamine synthesis in relation to dopamine D₂ receptor occupancy and the resulting changes in dopamine synthesis capacity in healthy men. On separate days, PET studies with [¹¹C]raclopride and L-[β-¹¹C]DOPA were performed under resting condition and with single doses of aripiprazole given orally. Occupancy of dopamine D₂ receptors corresponded to the doses of aripiprazole, but the changes in dopamine synthesis capacity were not significant, nor was the relation between dopamine D₂ receptor occupancy and these changes. A significant negative correlation was observed between baseline dopamine synthesis capacity and changes in dopamine synthesis capacity by aripiprazole, indicating that this antipsychotic appears to stabilize dopamine synthesis capacity. The therapeutic effects of aripiprazole in schizophrenia might be related to such stabilizing effects on dopaminergic neurotransmission responsivity.     

3,4-Dihydroxy- and 3,4-methylenedioxy- phenanthrene-type alkaloids with high selectivity for D2 dopamine receptor

Dopamine-mediated neurotransmission plays an important role in relevant psychiatric and neurological disorders. Nowadays, there is an enormous interest in the development of new drugs acting at the dopamine receptors (DR) as potential new targets for the treatment of schizophrenia or Parkinson’s disease. Previous studies have revealed that isoquinoline compounds such as tetrahydroisoquinolines (THIQs) can behave as selective D₂ dopaminergic alkaloids. In the present study we have synthesized five aporphine compounds and five phenanthrene alkaloids and evaluated their potential dopaminergic activity. Binding studies on rat striatal membranes were used to evaluate their affinity and selectivity towards D₁ and D₂ DR. Phenanthrene type alkaloids, in particular the 3,4-dihydroxy- and 3,4-methylenedioxy derivatives, displayed high selectivity towards D₂ DR. Therefore, they are potential candidates to be used in the treatment of schizophrenia (antagonists) or Parkinson’s disease (agonists) due to their scarce D₁ DR-associated side effects.     

Disrupted‐in‐schizophrenia 1 functional polymorphisms and D2/D3 receptor availability: A [11C]‐(+)‐PHNO imaging study

The disrupted‐in‐schizophrenia 1 (DISC1) protein has been implicated in a range of biological mechanisms underlying chronic mental disorders such as schizophrenia. Schizophrenia is associated with abnormal striatal dopamine signalling, and all antipsychotic drugs block striatal dopamine 2/3 receptors (D_2/3Rs). Importantly, the DISC1 protein directly interacts and forms a protein complex with the dopamine D₂ receptor (D₂R) that inhibits agonist‐induced D₂R internalisation. Moreover, animal studies have found large striatal increases in the proportion of D₂R receptors in a high affinity state (D₂^highR) in DISC1 rodent models. Here, we investigated the relationship between the three most common polymorphisms altering the amino‐acid sequence of the DISC1 protein (Ser704Cys (rs821616), Leu607Phe (rs6675281) and Arg264Gln (rs3738401)) and striatal D_2/3R availability in 41 healthy human volunteers, using [¹¹C]‐(+)‐PHNO positron emission tomography. We found no association between DISC1 polymorphisms and D_2/3R availability in the striatum and D₂R availability in the caudate and putamen. Therefore, despite a direct interaction between DISC1 and the D₂R, none of its main functional polymorphisms impact striatal D_2/3R binding potential, suggesting DISC1 variants act through other mechanisms.