Coupling between GPR143 and dopamine D2 receptor is required for selective potentiation of dopamine D2 receptor function by L-3,4-dihydroxyphenylalanine in the dorsal striatum.

Dopamine (DA) is involved in neurological and physiological functions such as motor control. L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of DA, is conventionally believed to be an inert amino acid precursor of DA, and its major therapeutic effects in Parkinson's disease (PD) are mediated through its conversion to DA. On the contrary, accumulating evidence suggests that L-DOPA itself is a neurotransmitter. We here show that L-DOPA potentiates DA D2 receptor (DRD2) signaling through GPR143, the gene product of X-linked ocular albinism 1, a G-protein-coupled receptor for L-DOPA. In Gpr143-gene-deficient (Gpr143-/y ) mice, quinpirole, a DRD2/DRD3 agonist, -induced hypolocomotion was attenuated compared to wild-type (WT) mice. Administration of non-effective dose of L-DOPA methyl ester augmented the quinpirole-induced hypolocomotion in WT mice but not in Gpr143-/y mice. In cells co-expressing GPR143 and DRD2, L-DOPA enhanced the interaction between GPR143 and DRD2 and augmented quinpirole-induced decrease in cAMP levels. This augmentation by L-DOPA was not observed in cells co-expressing GPR143 and DRD1 or DRD3. Chimeric analysis in which the domain of GPR143 was replaced with GPR37 revealed that GPR143 interacted with DRD2 at the fifth transmembrane domain. Intracerebroventricular administration of a peptide that disrupted the interaction mitigated quinpirole-induced behavioral changes in WT mice but not in Gpr143-/y mice. These findings provide evidence that coupling between GPR143 and DRD2 is required for selective DRD2 modulation by L-DOPA in the dorsal striatum.

Dietary supplementation with fish oil reverses high fat diet-induced enhanced sensitivity to the behavioral effects of quinpirole.

Consuming a high fat diet can lead to many negative health consequences, such as obesity, insulin resistance, and enhanced sensitivity to drugs acting on dopamine systems. It has recently been demonstrated that dietary supplementation with fish oil, which is rich in omega-3 fatty acids, can prevent this high fat diet-induced enhanced sensitivity to dopaminergic drugs from developing. However, it is not known whether fish oil supplementation can reverse this effect once it has already developed. To test the hypothesis that dietary supplementation with fish oil will reverse high fat diet-induced enhanced sensitivity to quinpirole, a dopamine D2/D3 receptor agonist, male Sprague-Dawley rats were fed either standard chow (17% kcal from fat), high fat chow (60% kcal from fat), standard chow, or high fat chow supplemented with 20% (w/w) fish oil. Body weight, food consumption, and sensitivity to quinpirole-induced (0.0032-0.32 mg/kg) penile erections were examined throughout the course of the experiment. Eating high fat chow enhanced sensitivity of rats to quinpirole-induced penile erections (i.e. resulted in a leftward shift of the ascending limb of the dose-response curve). Dietary supplementation with fish oil successfully treated this effect, as dose-response curves were not different for rats eating standard chow and rats eating high fat chow with fish oil. These results suggest that in addition to preventing the negative health consequences of eating a high fat diet, fish oil can also reverse some of these consequences once they have developed.

The Quinpirole Hypolocomotive Effects are Strain and Route of Administration Dependent in SHR and SLA16 Isogenic Rats.

The SHR and SLA16 inbred strains present behavioral differences in anxiety/emotionality that could be under the influence of dopaminergic neurotransmission. In order to investigate the role of D2 receptors in modulating such differences, an agonist (quinpirole) and an antagonist (haloperidol) of this receptor were administered, either via systemic injection (IP), or microinjected into the ventral area of the hippocampus (vHIP). Quinpirole and haloperidol IP decreased locomotor activity, only in SLA16 rats in the open-field (OF), and in both strains in the elevated plus-maze (EPM). Quinpirole also increased the preference for the aversive areas of the EPM. Quinpirole vHIP decreased locomotor activity in both strains. Haloperidol vHIP did not elicit behavioural changes and no differences in the levels of D2 receptors and of dopamine transporter in the hippocampus were found. Results indicate that systemic activation/blocking of D2 receptors caused a strain-dependent hypolocomotion, whereas activation of D2 receptors in the vHIP, but not D2 receptor antagonism, regardless of dose, decreased general locomotor activity in the two strains. Therefore, we suggest that genomic differences in the chromosome 4 can influence the locomotor activity regulated by the D2 dopaminergic receptor, especially in the vHIP.

Trace amine-associated receptor 1 agonists RO5263397 and RO5166017 attenuate quinpirole-induced yawning but not hypothermia in rats.

Increasing evidence suggests that trace amine-associated receptor 1 (TAAR1) is an important modulator of the dopaminergic system. Existing molecular evidence indicates that TAAR1 regulates dopamine levels through interactions with dopamine transporters and D2 receptors. However, investigations to date have not been exhaustive and other pathways may be involved. In this study, we used a well-described set of behaviors, quinpirole-induced yawning and hypothermia, to explore the potential interaction of TAAR1 and D3 receptors, which are members of the 'D2-like' dopamine receptor subfamily. Previous studies have shown that for D2/D3 receptor agonists, the induction of yawning is a D3 receptor-mediated effect, whereas the inhibition of yawning and induction of hypothermia are D2 receptor-mediated effects. Quinpirole produced an inverted U-shaped dose-effect curve for yawning, which was shifted downward dose-dependently by each of the TAAR1 agonists RO5263397 and RO5166017. Quinpirole also produced dose-dependent hypothermia, which was not affected by either TAAR1 agonist. These results suggest that TAAR1 agonists may interact with D3 receptors and/or its downstream pathways, as opposed to D2 receptors. These findings may shed light on a previously unexplored possibility for the mechanism of TAAR1-mediated effects.

Dietary supplementation with fish oil prevents high fat diet-induced enhancement of sensitivity to the behavioral effects of quinpirole.

Eating a diet high in fat can lead to negative health consequences, including obesity and insulin resistance. Omega-3 polyunsaturated fatty acids (such as those found in fish oil) prevent high fat diet-induced obesity and insulin resistance in rats. Eating a high fat diet also enhances sensitivity of rats to the behavioral effects of drugs that act on dopamine systems (e.g. quinpirole, a dopamine D2/D3 receptor agonist). To test the hypothesis that dietary supplementation with fish oil prevents high fat diet-induced enhanced sensitivity to the behavioral effects of quinpirole (0.0032-0.32 mg/kg), male rats ate standard laboratory chow, high fat chow, standard chow with fish oil, or high fat chow with fish oil (20% w/w). After 5 weeks, rats eating high fat chow were more sensitive (e.g. leftward shift of the quinpirole dose-response curve) than rats eating standard chow to yawning induced by quinpirole. Dietary supplementation with fish oil prevented this effect. That is, quinpirole dose-response curves were not different between rats eating high fat chow supplemented with fish oil and standard chow fed controls. These data add to a growing literature showing the complex relationship between diet and dopamine systems, and the health benefits of fish oil.

Early alterations of mitochondrial morphology in dopaminergic neurons from Parkinson's disease-like pathology and time-dependent neuroprotection with D2 receptor activation.

Neuroprotection, to prevent vulnerable cell populations from dying, is perhaps the main strategy for treating Parkinson's disease (PD). Yet in clinical practice, therapy is introduced after the disease is well established and many neurons have already disappeared, while experimentally, treatment is typically added at the same time that PD pathology is instigated. This study uses an already established Drosophila melanogaster model of PD to test for early markers of neurodegeneration and if those markers are reversible following neuroprotective treatment. Specifically, we treat primary neuronal cultures with the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) and track neuritic, dopaminergic mitochondria over time, observing a fragmenting change in their morphology before cell death. We then add a neuroprotective treatment (quinpirole, a D2 receptor agonist) at different timepoints to determine if the changes in mitochondrial morphology are reversible. We find that neuroprotective treatment must be added concomitantly to prevent changes in mitochondrial morphology and subsequent cell death. This work further supports Drosophila's use as a model organism and mitochondria's use as a biomarker for neurodegenerative disease. But mainly, this work highlights an import factor for experiments in neuroprotection - time of treatment. Our results highlight the problem that current neuroprotective treatments for PD may not be used the same way that they are tested experimentally.

Chronic activation of the D2 autoreceptor inhibits both glutamate and dopamine synapse formation and alters the intrinsic properties of mesencephalic dopamine neurons in vitro.

Dysfunctional dopamine (DA)-mediated signaling is implicated in several diseases including Parkinson's disease, schizophrenia and attention deficit and hyperactivity disorder. Chronic treatment with DA receptor agonists or antagonists is often used in pharmacotherapy, but the consequences of these treatments on DA neuron function are unclear. It was recently demonstrated that chronic D2 autoreceptor (D2R) activation in DA neurons decreases DA release and inhibits synapse formation. Given that DA neurons can establish synapses that release glutamate in addition to DA, we evaluated the synapse specificity of the functional and structural plasticity induced by chronic D2R activation. We show that chronic activation of the D2R with quinpirole in vitro caused a parallel decrease in the number of dopaminergic and glutamatergic axon terminals. The capacity of DA neurons to synthesize DA was not altered, as indicated by the lack of change in protein kinase A-mediated Ser(40) phosphorylation of tyrosine hydroxylase. However, the spontaneous firing rate of DA neurons was decreased and was associated with altered intrinsic properties as revealed by a prolonged latency to first spike after release from hyperpolarization. Moreover, D2R function was decreased after its chronic activation. Our results demonstrate that chronic activation of the D2R induces a complex neuronal reorganization involving the inhibition of both DA and glutamate synapse formation and an alteration in electrical activity, but not in DA synthesis. A better understanding of D2R-induced morphological and functional long-term plasticity may lead to improved pharmacotherapy of DA-related neurological and psychiatric disorders.

Pharmacologic neuroimaging of the ontogeny of dopamine receptor function.

Characterization of the ontogeny of the cerebral dopaminergic system is crucial for gaining a greater understanding of normal brain development and its alterations in response to drugs of abuse or conditions such as attention-deficit hyperactivity disorder. Pharmacological MRI (phMRI) was used to determine the response to dopamine transporter (DAT) blockers cocaine and methylphenidate (MPH), the dopamine releaser D-amphetamine (AMPH), the selective D1 agonist dihydrexidine, and the D2/D3 agonist quinpirole in young (<30 days old) and adult (>60 days old) rats. In adult rats, cocaine (0.5 mg/kg i.v.) or MPH (2 mg/kg) induced primarily positive cerebral blood volume (rCBV) changes in the dopaminergic circuitry, but negative rCBV changes in the young animals. Microdialysis measurements in the striatum showed that young rats have a smaller increase in extracellular dopamine in response to cocaine than adults. The young rats showed little rCBV response to the selective D1 agonist dihydrexidine in contrast to robust rCBV increases observed in the adults, whereas there was a similar negative rCBV response in the young and adult rats to the D2 agonist quinpirole. We also performed a meta-analysis of literature data on the development of D1 and D2 receptors and the DAT. These data suggest a predominance of D2-like over D1-like function between 20 and 30 days of age. These combined results suggested that the dopamine D1 receptor is functionally inhibited at young age.

GPR37 surface expression enhancement via N-terminal truncation or protein-protein interactions.

GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G-protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor's plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased the level of surface expression nearly as much as removal of the entire NT. In studies examining the effects of coexpression of GPR37 with a variety of other GPCRs, we observed significant increases in the level of GPR37 surface expression when the receptor was coexpressed with adenosine receptor A(2A)R or dopamine receptor D(2)R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D(2)R, resulting in modestly altered D(2)R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in the level of GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches (N-terminal truncation, coexpression with other receptors, and coexpression with syntenin-1) by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies with this orphan receptor.

Altered dopamine D2-like receptor binding in rats with behavioral sensitization to quinpirole: effects of pre-treatment with Ro 41-1049.

Repeated treatment with the dopamine D2/D3 receptor agonist quinpirole produces a sensitized behavioral response in rats manifested as an increase in locomotor activity. Pre-treatment with certain monoamine oxidase inhibitors, such as Ro 41-1049 [N-(2-aminomethyl)-5-(3-fluorophenyl)-4-thiazolecarboxamide HCl], changes the sensitized response from locomotion to stationary, self-directed mouthing. In this study, the effects of quinpirole sensitization, with and without pre-treatment with Ro 41-1049, were determined on dopamine D2-like receptors in the nucleus accumbens and the striatum. Long-Evans rats were pre-treated with Ro 41-1049 (1 mg/kg) 90 min prior to administration of quinpirole (0.5 mg/kg, 8 injections, every 3-4 days). Dopamine D2-like receptor binding was determined 3 days after the last injection by ex vivo radioligand assays using [3H]spiperone and [3H]quinpirole. Densities of [3H]spiperone- and [3H]quinpirole-labeled sites were both increased 32% in the nucleus accumbens of rats with demonstrated locomotor sensitization to quinpirole. In contrast, the density of dopamine D2-like receptors in quinpirole-sensitized rats pre-treated with Ro 41-1049 was not different from saline controls. These findings support the involvement of alterations in dopamine D2-like receptors in the development of locomotor sensitization to quinpirole and suggest that modification of these alterations in dopamine D2-like receptors contributes to the change from sensitized locomotion to mouthing observed when rats are pre-treated with Ro 41-1049.

Identification of neuroprotective compounds of caenorhabditis elegans dopaminergic neurons against 6-OHDA.

Parkinson's disease (PD) is a severe debilitating disorder, characterized by progressive and selective dopaminergic (DAergic) neuron degeneration within the substantia nigra pars compacta. Although current pharmacological treatments are effective in early stages of the disease, with time, most patients fail to respond to medications and develop serious motor complications. Therefore, devising novel and efficacious therapeutics that address not only the symptoms of PD, but also the cause, are of great importance. Unfortunately, many obstacles are associated with current PD research in mammalian-based systems, which limit the rate of progress. One solution is to investigate mechanisms of PD in model genetic organisms like Caenorhabditis elegans. In general, striking and profound similarities underlie the basic cellular and molecular processes between the worm and humans. The use of C. elegans over traditional mammalian-based systems holds the promise of an enhanced rate of discovery with lower associated costs. Here, we have utilized C. elegans to screen a variety of compounds, including specific dopamine (DA), GABA, and NMDA receptor agonists, as well as antagonists to identify those that protect against 6-OHDA-induced DAergic toxicity. Two DA D2 receptor agonists, bromocriptine and quinpirole, were found to protect against 6-OHDA toxicity in a dose-dependent manner. Surprisingly, these protective effects appear to involve receptor-independent mechanisms. Given the high degree of conservation of cellular processes between the worm and mammalian systems, these results are likely relevant and important toward understanding potentially novel mechanisms leading to DAergic neuroprotection in mammalian systems and, ultimately, new therapeutics for PD.

Nigrostriatal lesion induces D2-modulated phase-locked activity in the basal ganglia of rats.

There is a debate as to what modifications of neuronal activity underlie the clinical manifestations of Parkinson's disease and the efficacy of antiparkinsonian pharmacotherapy. Previous studies suggest that release of GABAergic striatopallidal neurons from D2 receptor-mediated inhibition allows spreading of cortical rhythms to the globus pallidus (GP) in rats with 6-hydroxydopamine-induced nigrostriatal lesions. Here this abnormal spreading was thoroughly investigated. In control urethane-anaesthetized rats most GP neurons were excited during the active part of cortical slow waves ('direct-phase' neurons). Two neuronal populations having opposite phase relationships with cortical and striatal activity coexisted in the GP of 6-hydroxydopamine-lesioned rats. 'Inverse-phase' GP units exhibited reduced firing coupled to striatal activation during slow waves, suggesting that this GP oscillation was driven by striatopallidal hyperactivity. Half of the pallidonigral neurons identified by antidromic stimulation exhibited inverse-phase activity. Therefore, spreading of inverse-phase oscillations through pallidonigral axons might contribute to the abnormal direct-phase cortical entrainment of basal ganglia output described previously. Systemic administration of the D2 agonist quinpirole to 6-hydroxydopamine-lesioned rats reduced GP inverse-phase coupling with slow waves, and this effect was reversed by the D2 antagonist eticlopride. Because striatopallidal hyperactivity was only slightly reduced by quinpirole, other mechanisms might have contributed to the effect of quinpirole on GP oscillations. These results suggest that antiparkinsonian efficacy may rely on other actions of D2 agonists on basal ganglia activity. However, abnormal slow rhythms may promote enduring changes in functional connectivity along the striatopallidal axis, contributing to D2 agonist-resistant clinical signs of parkinsonism.

Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors enhances heterodimer formation: a mechanism for receptor cross-talk?

Dopamine and endogenous cannabinoids display complex interactions in the basal ganglia. One possible level of interaction is between CB1 cannabinoid and D2 dopamine receptors. Here, we demonstrate that a regulated association of CB1 and D2 receptors profoundly alters CB1 signaling. This provides the first evidence that CB1/D2 receptor complexes exist, are dynamic, and are agonist-regulated with highest complex levels detected when both receptors are stimulated with subsaturating concentrations of agonist. The consequence of this interaction is a differential preference for signaling through a "nonpreferred" G protein. In this case, D2 receptor activation, simultaneously with CB1 receptor stimulation, results in the receptor complex coupling to G alpha s protein in preference to the expected G alpha i/o proteins. The result of this interaction is an increase in the second messenger cAMP, reversing an initial synergistic inhibition of adenylyl cyclase activity seen at subthreshold concentrations of cannabinoid agonist. Additionally, a pertussis toxin insensitive component in the activation of extracellular signal-regulated kinase (ERK) 1/2 kinases by the cannabinoid agonist CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] is revealed in cells stably expressing both CB1 and D2 receptors. Thus, concurrent receptor stimulation promotes a heterooligomeric receptor complex and an apparent shift of CB1 signaling from a pertussis toxin-sensitive inhibition to a partly pertussis toxin-insensitive stimulation of adenylyl cyclase and ERK 1/2 phosphorylation.

Analysis of RGSZ1 protein interaction with Galphai subunits.

RGSZ1 has been reported to interact with G-protein subunits of the Galphai family and function as a GTPase-accelerating protein on intrinsic Galphai GTPase activity. This article describes several experimental approaches and assays used to investigate the effect of RGSZ1 on Galphai subunits. The formats described here include physical and functional interaction assays by which the association of RGSZ1 with Galphai is explored both in vitro and in vivo. The methods analyzing physical interaction include pull-down and coimmunoprecipitation assays. We also apply yeast two-hybrid techniques to detect RGSZ1 protein interaction with Galpha subunits. Additionally, we developed several functional assay systems to identify the functional relationship between RGSZ1 and Galphai, such as the single turnover GTPase assay, yeast pheromone response assay, mitogen-activated protein kinase assay, and serum response element reporter assay.

Novel drug interactions at D(2) dopamine receptors: modulation of [3H]quinpirole binding by monoamine oxidase inhibitors.

D(2) dopamine receptors are the principal target of drugs used to treat schizophrenia and Parkinson's disease. Recent findings suggest novel drug interactions at D(2) receptors, specifically interactions of monoamine oxidase inhibitors (MAOIs) at a novel binding site that modulates the binding of [3H]quinpirole to the D(2) receptor. That MAOIs inhibit [3H]quinpirole binding challenges the traditional understanding of ligand interactions at dopamine receptors and may shed light on the mechanism of behavioral sensitization to psychostimulants and the pharmacology and toxicity of MAOIs.

Identification of the dopamine autoreceptor in the guinea-pig retina as D(2) receptor using novel subtype-selective antagonists.

1. Dopamine release in the retina is subject to modulation via autoreceptors, which belong to the D(2) receptor family (encompassing the D(2), D(3) and D(4) receptors). The aim of the present study was to determine the receptor subtype (D(2) vs D(3)) involved in the inhibition of dopamine release in guinea-pig retinal discs, using established (haloperidol, (S)-nafadotride) and novel dopamine receptor antagonists (ST-148, ST-198). 2. hD(2L) and hD(3) receptors were expressed in CHO cells and the pK(i) values determined in binding studies with [(125)I]-iodosulpride were: haloperidol 9.22 vs 8.54; ST-148 7.85 vs 6.60; (S)-nafadotride 8.52 vs 9.51; ST-198 6.14 vs 7.92. 3. The electrically evoked tritium overflow from retinal discs preincubated with [(3)H]-noradrenaline (which represents quasi-physiological dopamine release) was inhibited by the dopamine receptor agonists B-HT 920 (talipexole) and quinpirole (maximally by 82 and 71%; pEC(50) 5.80 and 5.83). The concentration-response curves of these agonists were shifted to the right by haloperidol (apparent pA(2) 8.69 and 8.23) and ST-148 (7.52 and 7.66). (S)-Nafadotride 0.01 microM and ST-198 0.32 microM did not affect the concentration-response curve of B-HT 920. 4. The dopamine autoreceptor in the guinea-pig retina can be classified as a D(2) receptor. ST-148 and ST-198 show an improved selectivity for D(2) and D(3) receptors when compared to haloperidol and (S)-nafadotride, respectively.

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.

Depressive behavior and alterations in receptors for dopamine and 5-hydroxytryptamine in the brain of the senescence accelerated mouse (SAM)-P10.

The senescence accelerated mouse (SAM) is known as a murine model of aging. SAM consists of senescence accelerated-prone mouse (SAMP) and senescence accelerated-resistant mouse (SAMR). Previous studies reported that SAMP10 exhibits age-related learning impairments and behavioral depression in a tail suspension test after 7 months. We investigated the changes in emotional behavior in a forced swimming test and in receptors for dopamine and 5-hydroxytryptamine (5-HT) in SAMP10. SAMP10 at 8 months showed an increase of immobility in the test compared with SAMR1. Treatment with desipramine (25 mg/kg, i.p., 3 days) in SAMP10 caused a decrease in immobility. In the cortex from SAMP10, [3H]quinpirole binding to D2/D3 dopamine receptors increased significantly compared with control SAMR1. In the hippocampus from SAMP10, [3H]8-hydroxy DPAT binding to 5-HT1A receptor increased. In midbrains from SAMP10, bindings of [3H]quinpirole and [3H]8-hydroxy DPAT increased. [3H]SCH23390 binding to D1/D5 receptors and [3H]ketanserin binding to 5-HT2 receptor in brain regions examined in SAMP10 were similar to those in SAMR1. The present findings represent the first neurochemical evidence of an increase of D2/D3 and 5-HT1A receptors in SAMP10. SAMP10 may be a useful model of aging associated depressive behavior.

Comparative pharmacological and functional analysis of the human dopamine D4.2 and D4.10 receptor variants.

The human dopamine D4 receptor is a D2-like receptor which is a target for most common neuroleptics. Previous investigations have shown that this receptor displays a large polymorphic variation in the third intracellular loop involving a variable number of direct imperfect tandem repeats (VNTR) of 16 amino acids. The shortest and longest repeat variants reported to date contain two and 10 repeat units (D4.2 and D4.10). No major pharmacological differences have been reported for the most common variants of this receptor (D4.2, D4.4 and D4.7), although the D4.7 was reported by us to display a slightly lower potency for dopamine in functional assays. Direct pharmacological and functional comparison of the longest and shortest variants in this study suggest no major discrepancies in pharmacological or functional profile between both receptors. Both receptors display, on average, a 15-fold and 90-fold lower potency for epinephrine and norepinephrine, respectively, compared with dopamine. We observed small increases in functional potency and affinity for dopamine and quinpirole at the D4.10 receptor variant compared with the D4.2 receptor. Our data indicate that there is no direct relationship between the length of the polymorphism and changes in pharmacology or functional activity. These findings are a suitable caution against the arbitrary pooling of D4 receptor VNTR genotypes in genetic studies, based on length.

Efficient functional coupling of the human D3 dopamine receptor to G(o) subtype of G proteins in SH-SY5Y cells.

1 The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines. 2 In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells. 4 More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.