Effect of amygdaloid kindling on rat striatal dopamine D1- and D2-receptors

The effect of kindling on dopaminergic (DA) neurotransmission was assessed by measuring dopamine D₁- and D₂-receptor binding in the dorsal and ventral striatum of rats either 2 hours (short-term) or 3–4 weeks (long-term) after the last kindled seizure. Kindling did not have any significant long-term effect on DA D₂-receptor K_d or B_max values in the dorsal or ventral striatum or on DA D₁-receptor parameters in the dorsal striatum. The short-term effect of kindled seizures was to abolish the asymmetry in DA D₂-receptor density observed in the dorsal striatum of control rats. DA D₁-receptor density was also increased in the dorsal striatum contralateral to the kindled amygdala of short-term rats. The short-term effects support the notion that limbic seizures can modify the lateral imbalance of DA activity in the striatum.     

Lack of effect of chronic desipramine treatment on dopaminergic activity in the nucleus accumbens of the rat

The binding of [³H]SCH 23390 to dopamine (DA) D₁-receptors was measured in the nucleus accumbens of rats treated chronically with desipramine for 14 days. DA D₁ — and D₂-receptor binding using [³H]SCH 23390 and [³H]spiperone, respectively as ligands, was determined in rats treated for 28 days. NeitherB _max norK _d values were influenced by chronic desipramine treatment. In addition, chronic desipramine treatment (28 days) did not influence the dose dependent, quinpirole (10–1000 nM)-mediated inhibition of the electrically stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens slices or the dose dependent increase in [³H]DA release and decrease in [¹⁴C]ACh release in the presence of 1 and 10 M nomifensine. Therefore, our results suggest that the effect of chronic antidepressant treatment cannot be attributed to changes in either DA D₁₁-or D₂-receptor binding or DA D₂-receptor function in the nucleus accumbens.     

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.     

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dopamine D₂ and D₄ receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D₂R–D_4.xR (D_4.2R, D_4.4R or D_4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET¹ techniques with the D₂R and D_4.7R receptors being the least effective in forming heteromers. Allosteric receptor–receptor interactions in D₂R–D_4.2R and D₂R–D_4.4 R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor–receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor–receptor interaction interfaces.     

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary 1. Our aim was to test the hypothesis that selectivity for D₃ dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with³H-emonapride, using human D₃ receptors in mouse fibroblasts and D₂ receptors in rat striatal tissue.3. D₃ receptors showed a picomolar affinity for³H-emonapride, Na⁺ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D₃/D₂ pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (–)-PPHT and its fluorescein derivative], (–)-N-propylnorapomorphine, (–)-3-PPP, (–)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D₃-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.     

Lack of interaction between α2 and dopamine D2-receptors in mediating their inhibitory effects on [3H]dopamine release from rat nucleus accumbens slices

The ₂-adrenoceptor agonist, UK14304, dose-dependently inhibited the electrically stimulated release of dopamine (DA) from rat nucleus accumbens slices. This effect was antagonized by idazoxan, confirming that it was an ₂-adrenoceptor mediated effect. There was no evidence of endogenous activation of noradrenergic receptors suggesting that the ₂-adrenoceptor agonist was not acting presynaptically to inhibit noradrenaline release. An in vitro superfusion technique was used to investigate wheher there was any interaction between ₂-adrenoceptors and DA D₂-receptors in mediating their inhibitory effects on [³H]DA release from rat nucleus accumbens slices. ₂-Adrenoceptor and DA D₂-receptors interact with similar second messenger systems and it was considered that they may compete for a common pool of G-proteins. The inhibitory effects of the ₂-adrenoceptor agonist, UK14304, and the DA receptor agonists, quinpirole, apomorphine and pergolide were not independent. However, there was no evidence of any interaction between UK14304 and the DA D₂-receptor antagonists, sulpiride or haloperidol, suggesting that the two receptors do not compete for a common pool of G-proteins in mediating their inhibitory effects on DA release.     

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.     

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.     

GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the -amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experiments to explore whether an activation of the GABA_A receptor could affect the binding characteristics of the D₂ DA receptor in membrane preparations of the rat neostriatum. The results show that GABA (30–100 nM) significantly increases the dissociation constant of the high affinity (K_H) D₂ DA binding site (labelled with the selective D₂ DA receptor antagonist [³H]raclopride and that such an effect is fully counteracted by the GABA_A receptor antagonist bicuculline (1 M). It is suggested that such putative GABA_A/D₂ receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D₂ receptors.     

Roles of G protein and beta-arrestin in dopamine D2 receptor-mediated ERK activation

ERK activation by dopamine D₂ receptor (D₂R) has been extensively characterized in various cell types including brain tissues. However, the involvement of β-arrestin in the D₂R-mediated ERK activation is not clear yet. Three different strategies were employed in this study to determine the roles of G protein or β-arrestin in D₂R-mediated ERK activation. The cellular level of β-arrestins was reduced by RNA interference and pertussis toxin-insensitive Gi proteins were used to identify the G protein involved. Finally point mutations of D₂R in which coupling with G protein was abolished but the interaction with β-arrestin was increased, were employed to determine whether the affinity between D₂R and β-arrestin is a critical factor for β-arrestin-mediated ERK activation. Our results show that G_i2 protein is involved in D₂R-mediated ERK activation but β-arrestins are either not involved or play minor role.     

Effect of exercise on hyperactivity,impulsivity and dopamine D2 receptor expression in the substantia nigra and striatum of spontaneous hypertensive rats

[Purpose]

Attention-deficit/hyperactivity disorder (ADHD) is a heritable, chronic, neurobehavioral disorder that is characterized by hyperactivity, inattention, and impulsivity. It is commonly believed that the symptoms of ADHD are closely associated with hypo-function of the dopamine system. Dopamine D₂ receptor activation decreases the excitability of dopamine neurons, as well as the release of dopamine. Physical exercise is known to improve structural and functional impairments in neuropsychiatric disorders. We investigated the therapeutic effect of exercise on ADHD.

[Methods]

Open field task and elevated-plus maze task were used in the evaluation of hyperactivity and impulsivity, respectively. Dopamine D₂ receptor expression in the substantia nigra and striatum were evaluated by western blotting.

[Results]

The present results indicated that ADHD rats showed hyperactivity and impulsivity. Dopamine D₂ receptor expression in the substantia nigra and striatum were increased in ADHD rats. Exercise alleviated hyperactivity and impulsivity in ADHD rats. Furthermore, dopamine D₂ receptor expression in ADHD rats was also decreased by exercise.

[Conclusion]

We thus showed that exercise effectively alleviates ADHD-induced symptoms through enhancing dopamine D₂ expression in the brain.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.     

Adenylyl Cyclase Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs

1.The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D_2S, D_2L, D_3S, D_3L, and D₄) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity.2.COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the subunit of Gz. PTX treatment was employed to inactivate endogenous i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells.3.D_2S or D_2L receptors can couple to the same extent to Gi and to Gz. The D₄ dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D_3S and D_3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively.4.We have demonstrated for the first time that the two D₃ receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX.5.Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.     

Dopamine D2 and 5-hydroxytryptamine 5-HT(₂A) receptors assemble into functionally interacting heteromers

In view of the co-distribution of dopamine D_2LR and 5-hydroxytryptamine 5-HT_2A receptors (D_2LR and 5-HT_2AR, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D_2LR-5-HT_2AR heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D_2LR and the 5-HT_2AR form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D_2LR-5-HT_2AR heteromeric signaling was analyzed we found that the 5-HT_2AR-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D_2LR as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D_2LR-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D_2LR and 5-HT_2AR within the heteromer led to inhibition of the D_2LR functioning, thus suggesting the existence of a 5-HT_2AR-mediated D_2LR trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D₂R-5-HT_2AR may be involved in the receptor interface. Overall, the presence of the D_2LR-5-HT_2AR heteromer in discrete brain regions is postulated based on the existence of D_2LR-5-HT_2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Prenatal cocaine exposure revealed minimal postnatal changes in rat striatal dopamine D2 receptor sites and mRNA levels in the offspring

It has been reported from this laboratory that prenatal cocaine exposure results in the postnatal transient alterations of rat striatal dopamine uptake sites examined from postnatal 0–32 wk. The present study aims to examine whether this will result in a direct/indirect stimulation of dopamine D₂ receptors. Pregnant rats were dosed orally with cocaine hydrochloride (60 mg/kg/d) from gestational day (GD) 7–21. Control animals received an equivalent volume of water. The striatum from the offspring at postnatal 0–32 wk was examined. The radioligand [³H]sulpiride was used for the Scatchard analysis of the D₂ receptors, and the changes in the levels of mRNA for the D₂ receptor were studied using Northern blot analysis. Results from the present study revealed that in the control group, there was an age-dependent increase in the number of D₂ receptor sites (B _max:44.00±2.12 to 178.00±45.10 fmol/mg protein) and in the levels of D₂ mRNA from PN0–32 wk with the most rapid increase occurring during the first 4 wk of postnatal development. Prenatal cocaine exposure resulted in only a significant decrease (p<0.001) in the number of D₂ receptor sites at PN0 wk and in a 10% increase in mRNA levels at PN3, 4, and 12 wk. It was concluded from this study that prenatal cocaine exposure resulted in minimal postnatal changes in the dopamine D₂ receptor.     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D₃ metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D₃ peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D₃ [4β,25(OH)₂D₃] and was found at concentrations comparable to 1α,25(OH)₂D₃. Quantification of 1α,25(OH)₂D₃ in plasma required complete chromatographic separation of 1α,25(OH)₂D₃ from 4β,25(OH)₂D₃. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD₃, 24R,25(OH)₂D₃, 1α,25(OH)₂D₃, and 4β,25(OH)₂D₃ in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)₂D₃ as well as monitoring the newly identified circulating metabolite, 4β,25(OH)₂D₃.