Occupancy of Brain Dopamine D3 Receptors and Drug Craving: A Translational Approach

Selective dopamine D₃ receptor (D₃R) antagonists prevent reinstatement of drug-seeking behavior and decrease the rewarding effects of contextual cues associated with drug intake preclinically, suggesting that they may reduce drug craving in humans. GSK598809 is a selective D₃R antagonist recently progressed in Phase I trials. The aim of this study was to establish a model, based on the determination of the occupancy of brain D₃Rs (O^D₃R) across species, to predict the ability of GSK598809 to reduce nicotine-seeking behavior in humans, here assessed as cigarette craving in smokers. Using ex vivo [¹²⁵I](R)-trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([¹²⁵I]7OH-PIPAT) autoradiography and [¹¹C]PHNO positron emission tomography, we demonstrated a dose-dependent occupancy of the D₃Rs by GSK598809 in rat, baboon, and human brains. We also showed a direct relationship between O^D₃R and pharmacokinetic exposure, and potencies in line with the in vitro binding affinity. Likewise, GSK598809 dose dependently reduced the expression of nicotine-induced conditioned place preference (CPP) in rats, with an effect proportional to the exposure and O^D₃R at every time point, and 100% effect at O^D₃R values ⩾72%. In humans, a single dose of GSK598809, giving submaximal levels (72–89%) of O^D₃R, transiently alleviated craving in smokers after overnight abstinence. These data suggest that either higher O^D₃R is required for a full effect in humans or that nicotine-seeking behavior in CPP rats only partially translates into craving for cigarettes in short-term abstinent smokers. In addition, they provide the first clinical evidence of potential efficacy of a selective D₃R antagonist for the treatment of substance-use disorders.     

Reduced Levels of Serotonin 2A Receptors Underlie Resistance of Egr3-Deficient Mice to Locomotor Suppression by Clozapine

The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients'' brains. We have previously reported that Egr3-deficient (Egr3^−/−) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3^−/− mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3^−/− and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT_2AR) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3^−/− mice. Radioligand binding revealed nearly 70% reduction in 5HT_2AR expression in the prefrontal cortex of Egr3^−/− mice compared with controls. Egr3^−/− mice also exhibit a decreased head-twitch response to 5HT_2AR agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3^−/− mice to the locomotor suppressive effects of SGAs, and suggest that 5HT_2ARs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT_2AR in Egr3^−/− mice aligns with numerous studies reporting decreased 5HT_2AR levels in the brains of schizophrenia patients, and the gene encoding the 5HT_2AR is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.     

The effects of benzamide analogues on cocaine self-administration in rhesus monkeys

Rationale: Based on the differential distribution of dopamine (DA) D₃ receptors in mesolimbic regions relative to nigrostriatal regions, the hypothesis was that D₃-selective antagonists (i.e., higher affinity at D₃- than D₂-receptors) would be more potent than D₂-selective antagonists at decreasing total cocaine intake relative to disrupting rates of responding. Objective: To evaluate the effects of acute administration of seven DA antagonists with varying affinities for D₂ and D₃ receptors in monkeys self-administering cocaine. Methods: Rhesus monkeys were trained to self-administer intravenous cocaine (0.01–0.3 mg/kg per injection) under a fixed-interval (FI) 5-min schedule during daily 4-h sessions. The use of a FI schedule allowed for independent assessment of rate effects and changes in reinforcement frequency as a consequence of drug pretreatments. The compounds examined, in order of D₃ binding affinity, were: 2,3-dimethoxy-N-(9-p-fluorobenzyl)-azabicyclo[3.3.1]nonan-3β-yl benzamide (MABN) = eticlopride = 5-bromo- 2,3-dimethoxy-N-[1-(4-fluorobenzyl)piperidin-4-yl]benz-amide (BBP) > spiperone > fluoroclebopride (FCP) > 2,3-dimethoxy-N-(p-fluorobenzyl)piperdin-4-yl benzamide (MBP) > haloperidol. Results: In the absence of any pretreatments, cocaine-maintained responding varied as a function of dose and was characterized as an inverted U-shaped function, while cocaine intake increased in a dose-related fashion. When the dose of cocaine that maintained peak rates was available, all DA antagonists decreased response rates and cocaine intake in a dose- dependent manner. Increases in cocaine dose attenuated the effects of the DA antagonists, resulting in rightward shifts of the cocaine dose–response curves. Based on the ratio of behavioral potency at decreasing response rates relative to intake (ED₅₀ rate/ED₅₀ intake) when the highest cocaine dose was available, the order of potency and ED₅₀ ratio values were: MABN (2.5) > eticlopride (1.63) > BBP = spiperone (1.5) > FCP (1.35) > MBP = haloperidol (0.89). This order parallels each compound’s affinity at D₃ receptors (r ²=0.84) to a greater degree than D₂ receptor affinity (r ²=0.34). Conclusions: These results, using a FI schedule of cocaine self-administration, suggest that D₃ receptor antagonists are more likely to selectively decrease intake relative to response rates than D₂ receptor antagonists. Received: 8 January 1999 / Final version: 15 June 1999     

Locomotor and discriminative-stimulus effects of cocaine in dopamine D<Subscript>5</Subscript> receptor knockout mice

Rationale Dopamine D₁-like antagonists block several effects of cocaine, including its locomotor-stimulant and discriminative-stimulus effects. Because these compounds generally lack selectivity among the dopamine D₁ and D₅ receptors, the specific roles of the subtypes have not been determined. Objectives Dopamine D₅ receptor knockout (DA D₅R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D₅ dopamine receptors in the effects of cocaine. In addition, effects of the D₁-like antagonist, SCH 39166 were also studied to further clarify the roles of D₁ and D₅ dopamine receptors in the discriminative-stimulus effects of cocaine. Methods DA D₅R KO, HET and WT mice were treated with cocaine (3–30 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a two-lever food-reinforcement (FR10) procedure. Doses of cocaine (1.0–10 mg/kg) were administered 5 min before 15-min test-sessions. Results Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D₅R WT mice. Both DA D₅R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; doses of 1.0–10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. SCH 39166, at inactive to fully active doses (0.01–0.1 mg/kg) produced predominately saline-appropriate responding. SCH 39166 produced a dose-dependent rightward shift in the cocaine dose-effect curve in all genotypes, with similar apparent affinities. Conclusions The present data suggest an involvement of DA D₅R in the locomotor stimulant effects of cocaine. In addition, the data indicate that there is little involvement of the DA D₅R in the discriminative-stimulus effects of cocaine. In addition, the antagonism data suggest a role of the D₁ receptor in the behavioral effects of cocaine.     

Chronic Δ9-Tetrahydrocannabinol Exposure Induces a Sensitization of Dopamine D2/3 Receptors in the Mesoaccumbens and Nigrostriatal Systems

Δ⁹-Tetrahydrocannabinol (THC), through its action on cannabinoid type-1 receptor (CB₁R), is known to activate dopamine (DA) neurotransmission. Functional evidence of a direct antagonistic interaction between CB₁R and DA D₂-receptors (D₂R) suggests that D₂R may be an important target for the modulation of DA neurotransmission by THC. The current study evaluated, in rodents, the effects of chronic exposure to THC (1 mg/kg/day; 21 days) on D₂R and D₃R availabilities using the D₂R-prefering antagonist and the D₃R-preferring agonist radiotracers [¹⁸F]fallypride and [³H]-(+)-PHNO, respectively. At 24 h after the last THC dose, D₂R and D₃R densities were significantly increased in midbrain. In caudate/putamen (CPu), THC exposure was associated with increased densities of D₂R with no change in D₂R mRNA expression, whereas in nucleus accumbens (NAcc) both D₃R binding and mRNA levels were upregulated. These receptor changes, which were completely reversed in CPu but only partially reversed in NAcc and midbrain at 1 week after THC cessation, correlated with an increased functionality of D_2/3R in vivo, based on findings of increased locomotor suppressive effect of a presynaptic dose and enhanced locomotor activation produced by a postsynaptic dose of quinpirole. Concomitantly, the observations of a decreased gene expression of tyrosine hydroxylase in midbrain together with a blunted psychomotor response to amphetamine concurred to indicate a diminished presynaptic DA function following THC. These findings indicate that the early period following THC treatment cessation is associated with altered presynaptic D_2/3R controlling DA synthesis and release in midbrain, with the concurrent development of postsynaptic D_2/3R supersensitivity in NAcc and CPu. Such D_2/3R neuroadaptations may contribute to the reinforcing and habit-forming properties of THC.     

Dopamine D2-Like Receptors and Behavioral Economics of Food Reinforcement

Previous studies suggest dopamine (DA) D₂-like receptor involvement in the reinforcing effects of food. To determine contributions of the three D₂-like receptor subtypes, knockout (KO) mice completely lacking DA D₂, D₃, or D₄ receptors (D₂R, D₃R, or D₄R KO mice) and their wild-type (WT) littermates were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) plotted to assess elasticity of demand. Under both economies, as price increased, food pellets obtained decreased more rapidly (ie, food demand was more elastic) in DA D₂R KO mice compared with WT littermates. Extinction of responding was studied in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction. Extinction in DA D₂R KO mice occurred less rapidly compared with WT mice in both contexts. Elasticity of food demand was higher in DA D₄R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D₄R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D₃R KO mice and WT littermates. These results indicate that the D₂R is the primary DA D₂-like receptor subtype mediating the reinforcing effectiveness of food.     

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent,clozapine, in the rat

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat.
2. The close structural analogues of clozapine, loxapine (0.1 mg kg⁻¹ s.c.) and iso-clozapine (1 and 3 mg kg⁻¹ s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg⁻¹ s.c.) did not produce catalepsy, but at a dose of 1 mg kg⁻¹ significantly inhibited catalepsy induced by loxapine (0.3 mg kg⁻¹ s.c.).
3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D₂/5-HT_1A, D₂/5-HT_1B/1D and D₂/α₂-receptor affinity (K_D) ratios: i.e. 30–100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D₂ to 5-HT_2A, 5-HT_2C or D₁ did not reflect the grouping of cataleptic and non-cataleptic compounds.
4. Co-treatment with the α₂-adrenoceptor antagonists, yohimbine (1–10 mg kg⁻¹ s.c.), RX 821002 (1–10 mg kg⁻¹ s.c.) and MK-912 (0.3 and 1 mg kg⁻¹ s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg⁻¹). Yohimbine (1–10 mg kg⁻¹ s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg⁻¹ s.c.). The α₂-adrenoceptor antagonists had no effect per se.
5. Neither yohimbine (10 mg kg⁻¹) nor RX821002 (3 mg kg⁻¹) altered the cataleptic response to the D₁ receptor antagonist, SCH 23390 (1 mg kg⁻¹ s.c.), while, like clozapine, both compounds abolished the response to the 5-HT_2A receptor antagonist, MDL 100,151 (3 mg kg⁻¹ s.c.).
6. The present data strongly implicate α₂-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

     

Dopamine-₃ receptor modulates intraocular pressure: implications for glaucoma

The aim of the present study was to investigate the role of D₃ receptor on intraocular pressure regulation using WT and KO D₃R^?/? mice. Both mice were used with normal eye pressure or steroid-induced ocular hypertension. As measured by tonometry, the topical application of 7-OH-DPAT, a dopamine D₃-preferring receptor agonist, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice both in an ocular normotensive group and an ocular hypertensive group. Pretreatment with U-99194A, a D₃ receptor antagonist, reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of intraocular pressure was observed after topical application of 7-OH-DPAT in KO D₃R^?/? mice. PCR analysis demonstrated the presence of all dopamine receptor genes in eye tissues obtained from WT mice, and the lack of D₃R mRNAs in KO mice. The present study identified the D₃R subtype as the most important receptor of the dopaminergic system to modulate intraocular pressure with relevant implications for glaucoma that represents one of the most crippling optic neuropathies.     

??-Amphetamine and Antipsychotic Drug Effects on Latent Inhibition in Mice Lacking Dopamine D2 Receptors

Drugs that induce psychosis, such as 𝒟-amphetamine (AMP), and those that alleviate it, such as antipsychotics, are suggested to exert behavioral effects via dopamine receptor D₂ (D₂). All antipsychotic drugs are D₂ antagonists, but D₂ antagonism underlies the severe and debilitating side effects of these drugs; it is therefore important to know whether D₂ is necessary for their behavioral effects. Using D₂-null mice (Drd₂−/−), we first investigated whether D₂ is required for AMP disruption of latent inhibition (LI). LI is a process of learning to ignore irrelevant stimuli. Disruption of LI by AMP models impaired attention and abnormal salience allocation consequent to dysregulated dopamine relevant to schizophrenia. AMP disruption of LI was seen in both wild-type (WT) and Drd₂−/−. This was in contrast to AMP-induced locomotor hyperactivity, which was reduced in Drd₂−/−. AMP disruption of LI was attenuated in mice lacking dopamine receptor D₁ (Drd₁−/−), suggesting that D₁ may play a role in AMP disruption of LI. Further supporting this possibility, we found that D₁ antagonist {"type":"entrez-protein","attrs":{"text":"SKF83566","term_id":"1157390490","term_text":"SKF83566"}}SKF83566 attenuated AMP disruption of LI in WT. Remarkably, both haloperidol and clozapine attenuated AMP disruption of LI in Drd₂−/−. This demonstrates that antipsychotic drugs can attenuate AMP disruption of learning to ignore irrelevant stimuli in the absence of D₂ receptors. Data suggest that D₂ is not essential either for AMP to disrupt or for antipsychotic drugs to reverse AMP disruption of learning to ignore irrelevant stimuli and further that D₁ merits investigation in the mediation of AMP disruption of these processes.     

Enhanced effect of neuropeptide Y on food intake caused by blockade of the V1A vasopressin receptor

Food intake is regulated by various factors such as neuropeptide Y. Neuropeptide Y potently induces an increase in food intake, and simultaneously stimulates arginine-vasopressin (AVP) secretion in the brain. Recently, we reported that V_1A vasopressin receptor-deficient (V_1AR^−/−) mice exhibited altered daily food intake accompanied with hyperglycemia and hyperleptinemia. Here, we further study the involvement of the AVP/V_1A receptor in the appetite regulation of neuropeptide Y with V_1AR^−/− mice and antagonists for the AVP receptor. The intra-cerebral-ventricle administration of neuropeptide Y induced greater food consumption in V_1AR^−/− mice than wild-type (WT) mice, whereas an anorexigenic effect of leptin was not different between the two groups. This finding suggests that the orexigenic effect of neuropeptide Y was enhanced in V_1AR^−/− mice, leading to the increased food intake in response to the neuropeptide Y stimulation. In addition, the neuropeptide Y-induced orexigenic effect was enhanced by co-administration of OPC-21268, an antagonist for the V_1A vasopressin receptor, into the cerebral ventricle in WT mice, whereas the neuropeptide Y-induced orexigenic effect was not affected by co-administration of SSR-149415, an antagonist for the V_1B vasopressin receptor. These results indicate that AVP could suppress the neuropeptide Y-induced orexigenic effect via the V_1A vasopressin receptor, and that blockade or inhibition of the AVP/V_1A receptor signal resulted in the enhanced neuropeptide Y-induced orexigenic effect. Thus, we show that the AVP/V_1A receptor is involved in appetite regulation as an anorexigenic factor for the neuropeptide Y-induced orexigenic effect.     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

Cocaine-induced locomotor activity and cocaine discrimination in dopamine D<Subscript>4</Subscript> receptor mutant mice

Rationale Previous studies have found a role for dopamine D₂-like receptors in many of the behavioral effects of cocaine, including its stimulation of locomotor activity and interoceptive discriminative-stimulus effects. However, given the lack of selectivity of most of the available pharmacological tools among D₂, D₃ and D₄ dopamine receptors, the roles of these specific receptors remain unclear. Objectives The roles of specific dopamine D₄ receptors in the behavioral effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects were investigated using dopamine D₄ receptor knockout (DA D₄R KO) and wild-type (WT) mice. Methods The mice were trained in daily sessions to discriminate IP injections of saline from cocaine (10 mg/kg). Responses on one of two response keys intermittently produced a food pellet; one response was reinforced in sessions following cocaine injection (10 mg/kg), and the other response was reinforced in sessions following saline injection. Each 20th response produced a food pellet (fixed-ratio, or FR20 schedule of reinforcement). The dose-effects of cocaine and its interaction with the D₂-like antagonist, raclopride, were assessed. Horizontal locomotor activity was also assessed in each genotype. Results As previously shown), cocaine was a more potent stimulant of locomotor activity in the DA D₄R KO mice compared to WT littermate mice. In addition, cocaine was more potent in producing discriminative-stimulus effects in DA D₄R KO mice (ED₅₀ value=0.50 mg/kg) compared to their WT littermates (ED₅₀ value=2.6 mg/kg). Raclopride shifted the cocaine dose-effect curve in both DA D₄R KO and WT mice, though the shift was greater for the DA D₄R KO mice. Conclusions The present results on the stimulation of activity and interoceptive/subjective effects of cocaine are consistent with the previously reported disregulation of dopamine synthesis in DA D₄R KO mice, and further suggest a role of the DA D₄R in vulnerability to stimulant abuse.     

Transport of Dopamine at the Blood-Brain Barrier of the Guinea Pig: Inhibition by Psychotropic Drugs and Nicotine

Purpose. Transport of dopamine (DA) across the blood-brain barrier (BBB) was examined in guinea pigs. Methods. In situ brain perfusion (1–10 min), capillary depletion, and high pressure liquid chrbmatography (HPLC) were used. Results. There was a saturable DA influx into the brain with a K_M of 389 ± 55 nM, and a V_MAXof 1.95 ± 0.25 pmol/min/g of brain. The diffusion constant, K_D, was not significantly different from zero. About 0.5% of DA remained tightly bound to cerebral microvessels isolated from the perfused brain. DA influx into the brain was not altered by the monoamine oxidase-B (MAO-B) inhibitor pargyline (50 µM). HPLC analysis of perfused brain confirmed transport of intact DA, and no detectable increases in DA metabolites were observed. At perfusate concentrations of 500 nM, several dopaminergic receptor antagonists inhibited [³H]-DA (21 nM) influx; the percent inhibitions for the mixed D₁ and D₂ antagonists haloperidol and chlorpromazine, the D₁, antagonist SCH-23390, and the D₂ antagonist spiperone were 90%, 68%, 77%, and 50%, respectively. Brain perfusion with nicotine (500 nM) inhibited DA uptake by 86%. This nicotine effect was not altered by mecamylamine, but was partially prevented by the nicotinic receptor antagonist hexamethonium. Conclusions. a) A significant cerebrovascular permeability to intact DA is mediated by a MAO-B independent specific transport system at the BBB, b) this system could be inhibited by D₁, and D₂ DA receptor antagonists, and c) DA blood-to-brain transport was inhibited by nicotine.     

Regulation of RANTES and IL-8 production in normal human dermal fibroblasts by active vitamin D3 (tacalcitol)

1. The production of chemokines, RANTES and IL-8 in cultured human dermal fibroblasts and the effects of tacalcitol (1α,24(R)-dihydroxyvitamin D₃) were studied using an enzyme-linked immunosorbent assay.
2. In the unstimulated condition, RANTES and IL-8 were at a trace level in the culture supernatant. On stimulation with TNF-α alone for 24 h, RANTES and IL-8 production were induced. Tacalcitol suppressed RANTES and IL-8 production dose-dependently at concentrations between 10⁻¹² M and 10⁻⁷ M.
3. When the cells were treated with TNF-α and IFN-γ in combination, RANTES production was enhanced, but IL-8 production was not changed, compared to TNF-α-treated cells. Tacalcitol decreased IL-8 production dose-dependently as observed in the TNF-α-treated cells. On the other hand, RANTES production was enhanced by 10⁻¹¹ M and 10⁻¹⁰ M of tacalcitol, and dose-dependently suppressed by tacalcitol concentrations higher than 10⁻⁹ M.
4. Active vitamin D₃ compounds, betamethasone valerate and cyclosporin A were compared with respect to their effects on chemokine production. Three active vitamin D₃ compounds, tacalcitol, 1α,25-dihydroxyvitamin D₃ and MC903 (calcipotriol), inhibited the production of RANTES and IL-8, with very similar potencies. Betamethasone valerate also inhibited these chemokine productions, but with greater potency than active vitamin D₃ compounds. Cyclosporin A significantly stimulated RANTES production at 10⁻⁶ M and IL-8 production at 10⁻⁷ M and 10⁻⁶ M.
5. The results of this study suggest that active vitamin D₃ compounds exert some beneficial effects in the treatment of inflammatory skin diseases via regulation of the production of chemokines by dermal fibroblasts.

     

Chronic blockade of angiotensin AT1 receptors improves cardinal symptoms of metabolic syndrome in diet-induced obesity in rats

Background and Purpose

AT₁ receptor antagonists decrease body weight gain in models of murine obesity. However, fewer data are available concerning the anti-obesity effects of these antagonists, given as a treatment after obesity had been established.

Experimental Approach

In spontaneously hypertensive rats, obesity was established by cafeteria diet (CD) feeding for 19 weeks. Rats were then were treated with telmisartan (8 mg·kg⁻¹·d⁻¹) or amlodipine (10 mg·kg⁻¹·d⁻¹; serving as blood pressure control) or telmisartan + amlodipine (2 + 10 mg·kg⁻¹·d⁻¹; to control for dose-dependency) for 17 weeks. Rats receiving only chow (C_chow) or CD-fed rats treated with vehicle (C_CD) served as controls.

Key Results

The CD feeding induced obesity, hyperphagia, hyperlipidaemia, and leptin and insulin resistance. Telmisartan reduced the CD-induced increase in body weight and abdominal fat mass. Whereas energy intake was higher rather than lower, the respiratory ratio was lower. After telmisartan, leptin-induced energy intake was reduced and respiratory ratio was increased compared with C_CD rats. Telmisartan also decreased plasma levels of triglycerides, free fatty acids and low-density lipoprotein. Amlodipine alone or the combination telmisartan + amlodipine did not affect body weight and eating behaviour. Telmisartan, but not amlodipine and telmisartan + amlodipine, improved glucose utilization. The decrease in BP reduction was almost the same in all treatment groups.

Conclusions and Implications

Telmisartan exerted anti-obesity effects and restored leptin sensitivity, given as a treatment to rats with obesity. Such effects required high doses of telmisartan and were independent of the decrease in blood pressure.     

Independent mediation of unconditioned motor behavior by striatal D1 and D2 receptors in rats depleted of dopamine as neonates

The effects of systemic administration of DA receptor antagonists suggest that unconditioned motor behavior in rats depleted of DA as neonates continues to be dependent upon dopaminergic transmission, yet the specific contribution of D₁ and D₂ receptors to these behaviors has been altered. The purpose of the present study was to determine whether these depletion-induced receptor changes are occurring at the level of striatal DA terminals and their targets. The ability of bilateral intrastriatal injections (0.5 µl) of DA receptor antagonists to induce motoric deficits was determined in adult rats treated with vehicle or 6-OHDA (100 µg, intraventricular) on postnatal day 3. Administration of the D₁-like antagonist SCH 23390 (0.5–2.0 µg) or the D₂-like antagonist clebopride (1.0–4.0 µg) induced dose-dependent akinesia, catalepsy, and somatosensory neglect in vehicle-treated controls. In contrast, neither antagonist produced deficits in rats depleted of forebrain DA as neonates. However,combined administration of SCH 23390+clebopride induced similar akinesia, catalepsy, and somatosensory neglect in both controls and DA depleted animals. Animals depleted of DA were more sensitive than controls to the low doses of this combined D₁+D₂ antagonism. These results demonstrate that activation of striatal DA receptors remains necessary for unconditioned motor behavior in rats depleted of DA as neonates. However, the specific contributions of D₁- and D₂-like receptors to these behaviors differ between intact animals and those depleted of DA as neonates. The ability of endogenous DA acting at either D₁ or D₂ receptors to support spontaneous motor behavior in rats depleted of DA as neonates may contribute to their relative sparing from parkinsonian deficits.     

Effects of haloperidol and SCH 23390 on acoustic startle in animals depleted of dopamine as neonates: implications for neuropsychiatric syndromes

Animals depleted of dopamine (DA) in the neonatal period and tested in adulthood exhibit some similarities to patients with schizophrenia, including increased sensitivity to DA agonists, altered sensitivity to DA receptor antagonists, and abnormalities of the acoustic startle response (ASR). In this study, we examined the contributions of D₁-like and D₂-like DA receptors to ASR measures in animals depleted of DA as neonates. Male rat pups received intracerebroventricular injections of 6-hydroxydopamine (DA depleted) or its vehicle (controls) at 3 days of age. Animals underwent startle testing ad adults (60–75 days of age) after administration of DA antagonists (haloperidol: 0.1 or 0.3 mg/kg, SCH 23390: 0.01 or 0.05 mg/kg) with and without DA agonist administration (apomorphine 0.5 mg/kg). ASR amplitude and prepulse inhibition (PPI: percentage decrease in startle amplitude due to a low intensity prepulse) were measured. DA depleted animals showed increased ASR amplitude and reduced PPI compared to controls. Administration of D₁-like or D₂-like DA antagonists significantly reduced overall ASR and increased PPI in both control and DA depleted animals, with DA depleted animals showing a relatively greater sensitivity to the D₁-like antagonist SCH 23390. Findings are discussed in terms of the role of residual DA in mediating ASR phenomena in depleted animals, differences between D₁/D₂ DA receptor mediation of ASR compared to other behaviors in DA depleted animals, and potential implications for neuropsychiatric syndromes such as schizophrenia.     

In Vivo Ethanol Experience Increases D2 Autoinhibition in the Ventral Tegmental Area

Alcoholism is characterized by compulsive alcohol intake after a history of chronic consumption. A reduction in mesolimbic dopaminergic transmission observed during abstinence may contribute to the negative affective state that drives compulsive intake. Although previous in vivo recording studies in rodents have demonstrated profound decreases in the firing activity of ventral tegmental area (VTA) dopamine neurons after withdrawal from long-term ethanol exposure, the cellular mechanisms underlying this reduced activity are not well understood. Somatodendritic dopamine release within the VTA exerts powerful feedback inhibition of dopamine neuron activity via stimulation of D₂ autoreceptors and subsequent activation of G protein-gated inwardly rectifying K⁺ (GIRK) channels. Here, by performing patch-clamp recordings from putative dopamine neurons in the VTA of mouse brain slices, we show that D₂ receptor/GIRK-mediated inhibition becomes more potent and exhibits less desensitization after withdrawal from repeated in vivo ethanol exposure (2 g/kg, i.p., three times daily for 7 days). In contrast, GABA_B receptor/GIRK-mediated inhibition and its desensitization are not affected. Chelating cytosolic Ca²⁺ with BAPTA augments D₂ inhibition and suppresses its desensitization in control mice, while these effects of BAPTA are occluded in ethanol-treated mice. Furthermore, inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release and Ca²⁺/calmodulin-dependent protein kinase II are selectively involved in the desensitization of D₂, but not GABA_B, receptor signaling. Consistent with this, activation of metabotropic glutamate receptors that are coupled to IP₃ generation leads to cross-desensitization of D₂/GIRK-mediated responses. We propose that enhancement of D₂ receptor-mediated autoinhibition via attenuation of a Ca²⁺-dependent desensitization mechanism may contribute to the hypodopaminergic state during ethanol withdrawal.     

Dopamine D1 and D2 mediation of the discriminative stimulus properties ofd-amphetamine and cocaine

Evidence suggests that stimulants such asd-amphetamine and cocaine act presynaptically by increasing the amount of dopamine (DA) available to stimulate postsynaptic DA receptors. Since two subpopulations of DA receptors (D₁ and D₂) exist, we investigated the role of both of these receptor subtypes in mediating the internal state produced by these stimulants. Two groups of rats (N=8/group) were trained to discriminate intraperitoneal (IP) injections of eitherd-amphetamine (1 mg/kg) or cocaine (10 mg/kg) from saline in a two-lever, water-reinforced, drug discrimination task. After stable performance was established (i.e., more than 85% correct under each training condition), substitution and combination tests were conducted with selective D₁ and D₂ agonists and antagonists. The D₂ agonist quinpirole (0.0313–0.125 mg/kg) mimicked both stimulant cues while the D₁ agoinst SKF 38393 (5–20 mg/kg) substituted partially for cocaine but notd-amphetamine. Combination tests with DA antagonists indicated that both the D₁ antagonist SCH 23390 (0.0063–0.25 mg/kg) and the D₂ antagonist haloperidol (0.125–0.5 mg/kg) attenuated the effects of both stimulants; in addition, the substitution of cocaine (20 mg/kg) ford-amphetamine was blocked by both DA antagonists. The ability of both D₁ and D₂ antagonists to attenuate the stimulus effects ofd-amphetamine and cocaine raises the possibility that a synergistic (enabling) interaction between D₁ and D₂ receptors may modulate stimulant cues.     

Opposite effects of CCKB agonists in grooming behaviour in rats: further evidence for two CCKB subsites

1. The hypothesis of the existence of two CCK_B receptor subsites, CCK_B1 and CCK_B2 corresponding probably to different coupling states of CCK_B receptors, was studied by measuring grooming behaviour in rats.
2. The B1 receptor agonist, BC197 (300 μg kg⁻¹, i.p.) produced a 45–50% decrease in grooming activity, which was prevented by both the CCK_B receptor antagonists CI-988 (20 μg kg⁻¹ i.p.) and L-365,260 (200 μg kg⁻¹, i.p.).
3. In contrast, 3, 10 and 30 μg kg⁻¹, i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150–190%). This effect was prevented by previous injection of 75 μg kg⁻¹ of L-365,260 while higher doses (200 μg kg⁻¹, i.p.) produced only a partial antagonism. Moreover, CI-988 (20 μg kg⁻¹, i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 μg kg⁻¹ of CI-988 tended to suppress the increase of grooming induced by BC264.
4. The effects of BC264 were prevented by the D₁ receptor (SCH 23390) and D₂ receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems.
5. This study brings additional evidence for the existence of the two CCK_B receptor subsites and suggests that particular attention should be focused on the selectivity of CCK_B receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.