Regulation of dopamine D2 receptor expression in grass carp pituitary cells: a possible mechanism for dopaminergic modification of luteinizing hormone synthesis.

In many fishes, dopamine (DA) strongly inhibits luteinizing hormone (LH) release by direct action at the pituitary level. In this study, the effect of DA on LH synthesis was examined by detecting its ß-subunit mRNA level in immature grass carp pituitary cells. Results showed that DA inhibited LHß mRNA expression and its inhibition was antagonized by a DA D2 receptor (DRD2) antagonist, sulpiride, suggesting that DA inhibited LH synthesis via DRD2. This notion was further supported by the finding that the grass carp DRD2 (gcDRD2) immunoreactivity was observed in the proximal pars distalis of the pituitary in which gonadotrophs are distributed. Accordingly, a full-length cDNA for DRD2 was cloned from grass carp pituitary and it showed closer phylogenetic relationships to the DA D2 receptors compared with the D3 and D4 or D1-like receptors in other vertebrates. Besides brain, the expression of this receptor in pituitary was revealed by tissue distribution assay, implying the pituitary function of gcDRD2 in immature grass carp. In grass carp pituitary cells, gcDRD2 transcript level was stimulated by DA and this stimulation was blocked by sulpiride. However, hCG, a functional homolog of grass carp LH, was found to inhibit gcDRD2 mRNA expression, indicating an intrapituitary negative feedback of LH on gcDRD2 expression. In view of our observation that the DRD2 mediated the dopaminergic inhibition of LH synthesis, we speculate that the DA stimulation and LH suppression on gcDRD2 may reinforce or attenuate the DA inhibition on LH synthesis, respectively and this regulation of gcDRD2 may at least partially contribute to the steady state levels of LH mRNA in prepubertal grass carp.

Molecular cloning of bullfrog D2 dopamine receptor cDNA: Tissue distribution of three isoforms of D2 dopamine receptor mRNA.

The cDNA encoding D2 dopamine receptor was cloned from the distal lobe of the bullfrog pituitary. The deduced amino acid sequence of the bullfrog D2 dopamine receptor (bfD2A) spanned 444 amino acids and exhibited typical features of those of D2 dopamine receptors cloned in other animals to date. It showed a high similarity of 75-87% with rat, turkey, Xenopus and tilapia counterparts. Further analysis of nucleotide sequence of the cDNA revealed the presence of putative truncated D2 dopamine receptor isoforms, bfD2B and bfD2C, of which nucleotide sequences lacked 12 and 99 nucleotides of the coding region for bfD2A, respectively. The alignment analysis indicated that putative bfD2C isoform was close to D2(S) subtype cloned in mammals and birds, whereas bfD2A and putative bfD2B isoforms were close to mammalian and avian D2(L) subtype and homologous to two isoforms of Xenopus. This is the first report of the presence of mRNAs for two D2(L)-like isoforms and one D2(S)-like isoform in a single species. The amino acid sequence responsible for producing isoforms is present in the third intracellular loop, which has been shown to play an important role in the coupling with G protein. Accordingly, differences in the mode of coupling with G protein among three isoforms were suggested. The expression of three isoforms mRNA in organs and tissues was analyzed by RT-PCR. In the brain, pars distalis and pars neurointermedia, mRNAs for three isoforms were invariably expressed, whereas only putative bfD2C mRNA was expressed in peripheral organs and tissues.

D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD.

The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD.

Characterization of the desensitization properties of five dopamine receptor subtypes and alternatively spliced variants of dopamine D2 and D4 receptors.

Proper regulation of brain dopaminergic activity is essential for maintaining normal mental functions. In this study, the regulatory properties of five different dopamine receptor subtypes and alternative splicing variants of dopamine D2 and D4 were examined. The stimulation of D1R, D2R, D5R but not D3R, D4R caused the robust translocation of beta-arrestin to the plasma membrane. When D1R or D3R were co-expressed with D2R, D1R significantly inhibited the sequestration of D2R, suggesting that the inhibitory effects of D1R on the D2R sequestration could explain the synergistic activity between two receptors. The sequestration of alternatively spliced isoforms of D2R was differently regulated by GRKs and beta-arrestins. Three alternative splicing variants of D4R produced a similar level of beta-arrestin translocation, and the studies with the deletion mutants of D4R within the third cytoplasmic loop revealed that the regions containing the SH3-binding domains are responsible for the beta-arrestin translocation.

Effects of 7-OH-DPAT and U 99194 on the behavioral response to hot plate test, in rats.

Aim of present study was to investigate in male Wistar rats, whether behavioral response to hot plate test application could be influenced by systemic administration of 7-OH-DPAT, a dopaminergic (DA) D3 versus D2 receptor agonist, or U 99194, a DA D3 versus D2 receptor antagonist. Each trial lasted no more than 10 s and the whole experimental session lasted 120 min. Animal behavior was recorded by means of a digital videocamera and later, frame by frame examined using a professional videorecorder. Latency of each behavioral pattern, characterizing the response, was analysed, showing significant changes only with U 99194. A multivariate cluster analysis indicated the presence of three main behavioral clusters (exploratory, primary responses to pain, escape) which, also, resulted significantly modified by both drugs. In addition, diagrams of preferential direction, obtained through multivariate stochastic analysis, evidenced switching probabilities differences among different patterns and clusters. Results demonstrate that the behavioral response to hot plate test application is altered following 7-OH-DPAT or U 99194 administration. These findings are discussed in terms of a) drugs influence on behavioral switching and learning processes; b) a likely activity on DA D3 versus D2 receptors.

The novel antidyskinetic drug sarizotan elicits different functional responses at human D2-like dopamine receptors.

Sarizotan (EMD 128130) is a chromane derivative that exhibits affinity at serotonin and dopamine receptors. Sarizotan effectively suppresses levodopa-induced dyskinesia in primate and rodent models of Parkinson's disease, and tardive dyskinesia in a rodent model. Results from clinical trials suggest that sarizotan significantly alleviates levodopa-induced dyskinesia. The functional effects of sarizotan on individual dopamine receptor subtypes are not known. Here we report the functional effects of sarizotan on human D2-like dopamine receptors (D2S, D2L, D3, D4.2 and D4.4) individually expressed in the AtT-20 neuroendocrine cell line. Using the coupling of D2-like dopamine receptors to G-protein coupled inward rectifier potassium channels we determined that sarizotan is a full agonist at D3 and D4.4 receptors (EC50=5.6 and 5.4 nM, respectively) but a partial agonist at D2S, D2L and D4.2 receptors (EC50=29, 23 and 4.5 nM, respectively). Consistent with its partial agonist property, sarizotan is an antagonist at D2S and D2L receptors (IC50=52 and 121 nM, respectively). Using the coupling of D2-like dopamine receptors to adenylyl cyclase we determined that sarizotan is a full agonist at D2L, D3, D4.2 and D4.4 receptors (EC50=0.51, 0.47, 0.48 and 0.23 nM, respectively) but a partial agonist at D2S receptors (EC50=0.6 nM).

Changes in food reward following smoking cessation: a pharmacogenetic investigation.

RATIONALE: Despite the high prevalence and public health significance of weight gain following smoking cessation, little is known about the underlying bio-behavioral mechanisms or effective therapies. OBJECTIVES: We evaluated the effects of bupropion on food reward following smoking abstinence and the moderating influence of genotype. METHODS: Seventy-one smokers of European ancestry were genotyped for the dopamine D2 receptor ( DRD2) Taq1 polymorphism and randomized to treatment with bupropion (300 mg) or placebo for smoking cessation. Subjects participated in two behavioral laboratory sessions during which the rewarding value of food was assessed using a behavioral economics measure: session 1 occurred prior to medication and before cessation of smoking; session 2 occurred following 3 weeks of medication and 1 week of sustained abstinence. RESULTS: Carriers of the DRD2 A1 minor allele exhibited significant increases in the rewarding value of food following abstinence from smoking, and these effects were attenuated by bupropion treatment ( P=0.03 for medication by genotype interaction). Further, higher levels of food reward at session 2 (post-quit) predicted a significant increase in weight by 6-month follow-up in the placebo group, but not in the bupropion-treated group ( P=0.006 for medication by food reward interaction). CONCLUSIONS: These results provide new evidence that the increase in body weight that occurs following smoking cessation is related to increases in food reward, and that food reward is partly determined by genetic factors. Bupropion's efficacy in attenuating abstinence-induced weight gain may be attributable, in part, to decreasing food reward.

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells.

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 GTPgammaS 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.

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene.

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.

Evolution of a D2 dopamine receptor intron within the great apes and humans.

Although direct DNA sequencing may allow rapid and high quality comparative phylogenetic analyses among species, such an approach may not be the most efficient method by which to make a large number of cross-species comparisons. We illustrate the use of Denaturing Gradient Gel Electrophoresis (DGGE) to screen a D2 Dopamine Receptor intron for DNA sequence variation, both within and between closely related species, in order to infer their evolutionary relationships. Our results suggest that: a) humans have less genetic variation than the great apes; b) pygmy chimpanzees have less genetic variation than common chimpanzees; and c) DNA sequence comparative analyses of primates require adequate sampling, both in number and in geographical range.

Dopaminergic systems. Dopamine receptors.

This article describes D1 and D2 receptor subfamilies, their neuroanatomic localizations and the possibility of their neuronal co-localization and relation to the other dopamine autoreceptors and those coupled to G-proteins as well as the actions of antipsychotic drugs on D1 and D2 receptor subfamilies are also analyzed.

Absence of D2S dopamine receptor in the prolactin-secreting MMQ pituitary clone: characterization of a wild D2L receptor coupled to native transduction mechanisms.

We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.

Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies.

Five or more dopamine receptor genes are expressed in brain. However, the pharmacological similarities of the encoded D1-D5 receptors have hindered studies of the localization and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised against recombinant D1 and D2 proteins and were shown to bind to the receptor subtypes specifically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the respective receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similar regional distributions in rat, monkey, and human brain, with the most intense staining in striatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions of each subtype were distinct and often complementary. D1 and D2 were differentially enriched in striatal patch and matrix compartments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat neostriatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat neostriatum. Presynaptic D1 and D2 receptors were also observed, indicating both subtypes may regulate neurotransmitter release. D1 was also present in axon terminals in the substantia nigra. These results provide a morphological substrate for understanding the pre- and postsynaptic functions of the genetically defined D1 and D2 receptors in discrete neuronal circuits in mammalian brain.

Unique binding characteristics of antipsychotic agents interacting with human dopamine D2A, D2B, and D3 receptors.

In the present study we have compared the pharmacological properties of human dopamine (DA) D2A, D2B, and D3 receptors expressed in mammalian cell lines, using [3H]raclopride as a radioligand. Most of the compounds tested had about equal affinity for D2A and D3 receptors, with the exception of remoxipride, which displayed a 10-fold D2 selectivity, and the aminotetralin (+)-UH 232, which displayed a 5-fold D3 selectivity. Several antipsychotic agents, including clozapine and substituted benzamides, bound with 2-3-fold higher to the D2B (short) than to the D2A (long) isoform, whereas others failed to differentiate between the two isoforms. The atypical antipsychotic agent clozapine bound in a biphasic manner and with unexpectedly high affinity (35 nM) to the D2B receptor, suggesting that clozapine may not be as D4 selective as reported previously. In addition, remoxipride, a new antipsychotic agent with low potential to produce extrapyramidal side effects, displayed 2-3-fold higher affinity for the D2B receptor than for the D2A receptor. Furthermore, sodium differently regulated clozapine and benzamide binding to the various DA receptor subtypes. Thus, sodium decreased the affinity of clozapine for D2A and D2B receptors about 3-fold, whereas the affinity for D3 receptors was unaltered. In contrast, the affinity of raclopride for the three DA receptor subtypes was increased by sodium. Whether the unique characteristics of the binding of clozapine and benzamides to cloned DA receptors demonstrated in the present study are related to the favorable clinical properties of these compounds remains to be elucidated.