Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

Dopamine receptors in human foetal brains:Characterization, regulation and ontogeny of [3H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [3H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC50 values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC50 values of 2.5 and 10 μM, respectively suggesting a D2 type receptor.Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 108 M−1 min−1 and 5.0 × 10−2 min−1, respectively. Equilibrium dissociation constant was 87 pM and the KD obtained by saturation binding was 73 pM.During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [3H]spiperone binding sites exhibited a heterogeneity with a high (KD, 13–85 pM) and a low (KD, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.GTP decreased the agonist affinity as observed by dopamine competition of [3H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC50 values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.

Dopamine receptors in a songbird brain.

Dopamine is a key neuromodulatory transmitter in the brain. It acts through dopamine receptors to affect changes in neural activity, gene expression, and behavior. In songbirds, dopamine is released into the striatal song nucleus Area X, and the levels depend on social contexts of undirected and directed singing. This differential release is associated with differential expression of activity-dependent genes, such as egr1 (avian zenk), which in mammalian brain are modulated by dopamine receptors. Here we cloned from zebra finch brain cDNAs of all avian dopamine receptors: the D1 (D1A, D1B, D1D) and D2 (D2, D3, D4) families. Comparative sequence analyses of predicted proteins revealed expected phylogenetic relationships, in which the D1 family exists as single exon and the D2 family exists as spliced exon genes. In both zebra finch and chicken, the D1A, D1B, and D2 receptors were highly expressed in the striatum, the D1D and D3 throughout the pallium and within the mesopallium, respectively, and the D4 mainly in the cerebellum. Furthermore, within the zebra finch, all receptors, except for D4, showed differential expression in song nuclei relative to the surrounding regions and developmentally regulated expression that decreased for most receptors during the sensory acquisition and sensorimotor phases of song learning. Within Area X, half of the cells expressed both D1A and D2 receptors, and a higher proportion of the D1A-only-containing neurons expressed egr1 during undirected but not during directed singing. Our findings are consistent with hypotheses that dopamine receptors may be involved in song development and social context-dependent behaviors.

Identification of dopamine receptors across the extant avian family tree and analysis with other clades uncovers a polyploid expansion among vertebrates.

Dopamine is an important central nervous system transmitter that functions through two classes of receptors (D1 and D2) to influence a diverse range of biological processes in vertebrates. With roles in regulating neural activity, behavior, and gene expression, there has been great interest in understanding the function and evolution dopamine and its receptors. In this study, we use a combination of sequence analyses, microsynteny analyses, and phylogenetic relationships to identify and characterize both the D1 (DRD1A, DRD1B, DRD1C, and DRD1E) and D2 (DRD2, DRD3, and DRD4) dopamine receptor gene families in 43 recently sequenced bird genomes representing the major ordinal lineages across the avian family tree. We show that the common ancestor of all birds possessed at least seven D1 and D2 receptors, followed by subsequent independent losses in some lineages of modern birds. Through comparisons with other vertebrate and invertebrate species we show that two of the D1 receptors, DRD1A and DRD1B, and two of the D2 receptors, DRD2 and DRD3, originated from a whole genome duplication event early in the vertebrate lineage, providing the first conclusive evidence of the origin of these highly conserved receptors. Our findings provide insight into the evolutionary development of an important modulatory component of the central nervous system in vertebrates, and will help further unravel the complex evolutionary and functional relationships among dopamine receptors.

Effect of Pyridoxine Deficiency in Young-Rats on High-Affinity Serotonin and Dopamine Receptors

The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors, [3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]- fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2 receptors in the corpus striatum were studied in pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant decrease in their binding affinities (Kd). However, there were no significant changes either in the maximal binding or binding affinity of striatal dopamine D- 1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the corresponding neurotransmitter concentrations in the pyridoxine-deficient rats.

Mechanisms of inverse agonist action at D-2 dopamine receptors

1 Mechanisms of inverse agonist action at the D-2(short) dopamine receptor have been examined. 2 Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [H-3]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. 3 Competition of inverse agonists versus [H-3] NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K-i values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K-coupled and K-uncoupled were statistically different for the set of compounds tested ( ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. 4 These observations were supported by simulations of these competition experiments according to the extended ternary complex model. 5 Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [S-35]GTPγ S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. 6 These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Oligomers of D-2 dopamine receptors - Evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands.

The influence of G protein subtype on agonist action at D-2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D-2 dopamine receptors to couple to G proteins and here we extend this work. The human D-2Short dopamine receptor and a natural polymorphism of this D-2Short(Ser(311)Cys), have been studied by co-expressing the receptors in insect cells with Gbeta(1)gamma(2) and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[H-3]spiperone competition binding experiments and through stimulation of [S-35]GTPgammaS binding. Although the Ser(311)Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D-2 receptor, agonist stimulation of [S-35]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins. (C) 2004 Elsevier Ltd. All rights reserved.

Mechanisms of agonist action at D-2 dopamine receptors

In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D-2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[S-35]thio) triphosphate ([S-35]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [S-35]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP. At both concentrations of [S-35]GTPgammaS, increasing GDP decreased the [S-35]GTPgammaS binding observed with maximally stimulating concentrations of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concentrations. Concentration-response experiments were performed for a range of agonists at the two [S-35]GTPgammaS concentrations and with different concentrations of GDP. At 0.1 nM [S-35]GTPgammaS, the potency of both full and partial agonists was dependent on the GDP concentration in the assays. At 10 nM [S-35]GTPgammaS, the potency of full agonists exhibited a greater dependence on the GDP concentration, whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [S-35]GTPgammaS concentration, the rate-determining step in G protein activation is the binding of [S-35]GTPgammaS to the G protein. At the higher [S-35]GTPgammaS concentration, for full agonists, [S-35]GTPgammaS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-determining.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Oligomers of D-2 dopamine receptors: evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands