The MCH1 receptor,an anti-obesity target,is allosterically inhibited by 8-methylquinoline derivatives possessing subnanomolar binding and long residence times

BACKGROUND AND PURPOSE

Melanin-concentrating hormone receptor 1 (MCH₁ receptor) antagonists are being considered as anti-obesity agents. The present study reports a new class of MCH₁ receptor antagonists with an 8-methylquinoline scaffold. The molecular mechanism of MCH₁ receptor blockade by these antagonists was examined.

EXPERIMENTAL APPROACH

The pharmacological properties of the 8-methylquinolines as exemplified by MQ1 were evaluated by use of multiple biophysical and cell-based functional assays.

KEY RESULTS

Multiple signalling pathways for Gα_i and Gα_q, and β-arrestin were inhibited by MQ1. Furthermore, MQ1 produced an insurmountable antagonism, causing a rightward shift of the curve for concentration-dependent binding of MCH along with a progressive reduction of the maximal response. The dissociation kinetics for MQ1 were determined from washout experiments as well as by affinity selection-MS. In short, MQ1 was shown to be a slowly dissociating reversible MCH₁ receptor blocker with a low K_off value.

CONCLUSION AND IMPLICATIONS

This is the first time that a slowly dissociating negative allosteric modulator of the MCH₁ receptor has been demonstrated to inhibit the numerous signalling pathways of this receptor. The characteristics of MQ1 are superior and distinct from previously reported MCH₁ receptor antagonists, making members of this chemotype attractive as drug candidates.     

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations

Background and purpose:

Current single drug treatments for rheumatoid arthritis have problems of limited efficacy and/or high toxicity. This study investigates the benefits of individual and combined treatments with dexamethasone and substance P and glutamate receptor antagonists in a rat model of arthritis.

Experimental approach:

Arthritis was induced in rats by unilateral intra-articular injection of Freund''s complete adjuvant. Separate groups of rats were subjected to the following treatments 15 min before induction of arthritis: (i) control with no drug treatment; (ii) single intra-articular injection of a NK₁ receptor antagonist RP67580; (iii) single intra-articular injection of a NMDA receptor antagonist AP7 plus a non-NMDA receptor antagonist CNQX; (iv) daily oral dexamethasone; and (v) combined treatment with dexamethasone and all of the above receptor antagonists. Knee joint allodynia, swelling, hyperaemia and histological changes were examined over a period of 7 days.

Key results:

Treatment with dexamethasone suppressed joint swelling, hyperaemia and histological changes that include polymorphonuclear cell infiltration, synovial tissue proliferation and cartilage erosion in the arthritic rat knees. Treatment with RP67580 or AP7 plus CNQX did not attenuate hyperaemia or histological changes, but reduced joint allodynia and swelling. Co-administration of dexamethasone with these receptor antagonists produced greater inhibition on joint allodynia and swelling than their individual effects.

Conclusions and implications:

The data suggest substance P and glutamate contribute to arthritic pain and joint swelling. The efficacy of dexamethasone in reducing arthritic pain and joint swelling can be improved by co-administration of substance P and glutamate receptor antagonists.     

A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents

BACKGROUND AND PURPOSE

Cannabinoid CB₁ receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects.

EXPERIMENTAL APPROACH

Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated.

KEY RESULTS

AM6545 binds to CB₁ receptors with a K_i of 1.7 nM and CB₂ receptors with a K_i of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB₁ receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB₁ receptor gene-deficient mice, but not in CB₁/CB₂ receptor double knockout mice.

CONCLUSIONS AND IMPLICATIONS

Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications.     

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.     

High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139

Aim:

To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules.

Methods:

Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies.

Results:

Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds.

Conclusion:

The findings provide important tools for further study of this orphan G-protein coupled receptor.     

Contractile effect of tachykinins on rabbit small intestine

Aim:

To study the role of the tachykinin receptors in spontaneous contractions of longitudinal and circular smooth muscle from rabbit small intestine and to determine the mechanism of action of Substance P (SP).

Methods:

Rabbit duodenum, jejunum and ileum segments were prepared. The spontaneous contractions of longitudinal and circular smooth muscle were recorded using a computer via an isometric force transducer. The specific agonists and antagonists of tachykinin receptors were added into the organ bath.

Results:

The agonists of tachykinin NK1 receptor (SP and [Sar9] SP), NK2 receptor (NKA and (β-Ala8)-NKA), and NK3 receptor (NKB and Senktide) all induced contractions in the small intestine. The contractions were diminished by NK1 receptor antagonist L-733,060, NK2 receptor antagonist GR-94800, and NK3 receptor antagonist SB 218795. Contractions caused by SP were also reduced by atropine, verapamil, PKC inhibitor staurosporine, and PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusion:

Ttachykinin NK1, NK2, and NK3 receptors mediate the contractions of the smooth muscle in rabbit intestine. Furthermore, SP acts directly on smooth muscle cells through the tachykinin NK1 receptor.     

Anti-obesity efficacy of LH-21, a cannabinoid CB(1) receptor antagonist with poor brain penetration, in diet-induced obese rats

BACKGROUND AND PURPOSE

Peripheral blockade of cannabinoid CB₁ receptors has been proposed as a safe and effective therapy against obesity, putatively devoid of the adverse psychiatric side effects of centrally acting CB₁ receptor antagonists. In this study we analysed the effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist with poor brain penetration, in an animal model of diet-induced obesity.

EXPERIMENTAL APPROACH

To induce obesity, male Wistar rats were fed a high-fat diet (HFD; 60 kcal% fat) whereas controls received a standard diet (SD; 10 kcal% fat). Following 10 weeks of feeding, animals received a daily i.p. injection of vehicle or 3 mg·kg⁻¹ LH-21 for 10 days. Plasma and liver samples were used for biochemical analyses whereas visceral fat-pad samples were analysed for lipid metabolism gene expression using real-time RT-PCR. In addition, the potential of LH-21 to interact with hepatic cytochrome P450 isoforms and cardiac human Ether-à-go-go Related Gene (hERG) channels was evaluated.

KEY RESULTS

LH-21 reduced feeding and body weight gain in HFD-fed animals compared with the control group fed SD. In adipose tissue, this effect was associated with decreased gene expression of: (i) leptin; (ii) lipogenic enzymes, including SCD-1; (iii) CB₁ receptors; and (iv) both PPARα and PPARγ. Although there were no significant differences in plasma parameters between HFD- and SD-fed rats, LH-21 did not seem to induce hepatic, cardiac or renal toxicity.

CONCLUSIONS AND IMPLICATIONS

These results support the hypothesis that treatment with the peripherally neutral acting CB₁ receptor antagonist, LH-21, may promote weight loss through modulation of visceral adipose tissue.     

A role for the sensory neuropeptide calcitonin gene-related peptide in endothelial cell proliferation in vivo

BACKGROUND AND PURPOSE

We have tested the hypothesis that calcitonin gene-related peptide (CGRP) is a mediator of capsaicin-induced angiogenesis in vivo.

EXPERIMENTAL APPROACH

In a series of experiments, the knee joints of rats were injected with CGRP, capsaicin or vehicle control. Groups of animals (n = 6) were treated with the CGRP receptor antagonist BIBN4096BS and/or the NK₁ receptor antagonist SR140333. Endothelium, proliferating endothelial cell nuclei and macrophages were identified 24 h later in the synovium by immunohistochemistry and quantified by image analysis. mRNA for the receptors for CGRP and adrenomedullin were sought in normal and inflamed rat and human synovia using RT-PCR.

KEY RESULTS

Intra-articular CGRP injection increased the endothelial cell proliferation index, whereas macrophage infiltration and knee joint diameters were similar to saline-injected controls. CGRP-induced endothelial cell proliferation was dose-dependently inhibited by BIBN4096BS. mRNA for adrenomedullin and the CGRP receptor subunits were detected in normal and inflamed human and rat synovia. In capsaicin-induced synovitis, the increased endothelial cell proliferation index was partially blocked by administration of NK₁ or CGRP antagonists individually and was reduced to the level of saline controls by coadministration of both receptor antagonists.

CONCLUSIONS AND IMPLICATIONS

These data support the hypothesis that CGRP stimulates angiogenesis in vivo directly by activating CGRP receptors. Capsaicin-induced endothelial cell proliferation was completely blocked by coadministration of CGRP and NK₁ receptor antagonists, indicating that both CGRP and substance P may contribute to angiogenesis in this model of synovitis.     

Pharmacological characterization of rat amylin receptors: implications for the identification of amylin receptor subtypes

BACKGROUND AND PURPOSE

Amylin (Amy) is an important glucoregulatory peptide and AMY receptors are clinical targets for diabetes and obesity. Human (h) AMY receptor subtypes are complexes of the calcitonin (CT) receptor with receptor activity-modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant receptor subtype, the elucidation of rat (r) AMY receptor pharmacology is necessary.

EXPERIMENTAL APPROACH

Receptors were transiently transfected into COS-7 cells and cAMP responses measured in response to different agonists, with or without antagonists. Competition binding experiments were performed to determine rAmy affinity.

KEY RESULTS

rCT was the most potent agonist of rCT_(a) receptors, whereas rAmy was most potent at rAMY_1(a) and rAMY_3(a) receptors. rAmy bound to these receptors with high affinity. Rat α-calcitonin gene-related peptide (CGRP) was equipotent to rAmy at both AMY receptors. Rat adrenomedullin (AM) and rAM2/intermedin activated all three receptors but were most effective at rAMY_3(a). AC187, AC413 and sCT_8-32 were potent antagonists at all three receptors. rαCGRP_8-37 displayed selectivity for rAMY receptors over rCT_(a) receptors. rAMY_8-37 was a weak antagonist but was more effective at rAMY_1(a) than rAMY_3(a).

CONCLUSIONS AND IMPLICATIONS

AMY receptors were generated by co-expression of rCT_(a) with rRAMP1 or 3, forming rAMY_1(a) and rAMY_3(a) receptors, respectively. CGRP was more potent at rAMY than at hAMY receptors. No antagonist tested was able to differentiate the rAMY receptor subtypes. The data emphasize the need for and provide a useful resource for developing new CT or AMY receptor ligands as pharmacological tools or potential clinical candidates.

LINKED ARTICLES

This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1     

Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells

Background and purpose:

The CCR5 chemokine receptor is a member of the G protein-coupled receptor (GPCR) family that is expressed by macrophages, memory T-lymphocytes and dendritic cells and is activated by chemotactic proteins (e.g. MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5]). CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus-1 (HIV-1) and some chemokines can inhibit HIV-1 infection by stimulating CCR5 receptor endocytosis. The aim of this study was to evaluate the effect of CCR5 antagonists on CCR5 endocytosis.

Experimental approach:

The effects of CCR5 agonists and antagonists on receptor internalization in CHO cells, expressing a C-terminal green fluorescent protein-tagged human CCR5 receptor (CCR5-GFP), were quantified using a confocal imaging plate reader.

Key results:

MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5] were all able to stimulate potently the internalization of CCR5-GFP. This effect was inhibited by the non-peptide antagonist TAK 779. The CCR5 peptide antagonist met-RANTES antagonized MIP-1α-mediated increases in intracellular free calcium but was also able to stimulate a substantial internalization of the human CCR5-GFP receptor. However, CHO cells exhibited an aminopeptidase activity that was able to metabolize sufficient met-RANTES into an agonist metabolite capable of stimulating calcium mobilization via CCR5 receptors in naïve cells.

Conclusions and implications:

These data suggest that there is an endogenous aminopeptidase activity on the surface of CHO cells, that produces a slow internalization of the receptor following a time-dependent conversion of receptor-bound met-RANTES from a CCR5 receptor antagonist into a CCR5 agonist molecule.     

Molecular requirements for inhibition of the chemokine receptor CCR8 – probe-dependent allosteric interactions

BACKGROUND AND PURPOSE

Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes.

EXPERIMENTAL APPROACH

The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα_i signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148.

KEY RESULTS

All compounds were highly potent inverse agonists with EC₅₀ values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC₅₀ values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [¹²⁵I]-CCL1 (K_i 3.4–842 nM), whereas the affinities measured against [¹²⁵I]-MC148 were less widely spread (K_i 0.37–27 nM), and matched the inverse agonist potencies.

CONCLUSION AND IMPLICATIONS

Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.     

CXCR3 antagonist VUF10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK–779

Background and Purpose

The chemokine receptor CXCR3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR3 antagonist VUF10085 and the broad spectrum antagonist TAK-779 which blocks CXCR3 along with CCR2 and CCR5.

Experimental Approach

Molecular modelling of CXCR3, followed by virtual ligand docking, highlighted several CXCR3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 (Asp-112^2:63), which was postulated to interact with the quaternary nitrogen of TAK-779. Validation of modelling was carried out by site-directed mutagenesis of CXCR3, followed by assays of cell surface expression, ligand binding and receptor activation.

Key Results

Mutation of Asn-132^3.33, Phe-207 and Tyr-271^6.51 within CXCR3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR3 conformation. Contrary to our hypothesis, mutation of Asp-112^2:63 had no observable effects on TAK-779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of Phe-131^3.32, Ile-279^6.59 and Tyr-308^7.43 were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK-779.

Conclusions and Implications

This more detailed definition of a binding pocket within CXCR3 for low MW antagonists should facilitate the rational design of newer CXCR3 antagonists, with obvious clinical potential.     

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B₂ receptor

BACKGROUND AND PURPOSE

Icatibant is a well-known kinin B₂ receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B₂ receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists.

EXPERIMENTAL APPROACH

Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B₂ receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B₂ receptors.

KEY RESULTS

Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B₂ receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B₂ receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B₂ receptor complex is proposed.

CONCLUSIONS AND IMPLICATIONS

MEN16132 dissociated from the B₂ receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.     

Neural Effects of Cannabinoid CB1 Neutral Antagonist Tetrahydrocannabivarin on Food Reward and Aversion in Healthy Volunteers

Background:

Disturbances in the regulation of reward and aversion in the brain may underlie disorders such as obesity and eating disorders. We previously showed that the cannabis receptor subtype (CB1) inverse agonist rimonabant, an antiobesity drug withdrawn due to depressogenic side effects, diminished neural reward responses yet increased aversive responses (Horder et al., 2010). Unlike rimonabant, tetrahydrocannabivarin is a neutral CB1 receptor antagonist (Pertwee, 2005) and may therefore produce different modulations of the neural reward system. We hypothesized that tetrahydrocannabivarin would, unlike rimonabant, leave intact neural reward responses but augment aversive responses.

Methods:

We used a within-subject, double-blind design. Twenty healthy volunteers received a single dose of tetrahydrocannabivarin (10mg) and placebo in randomized order on 2 separate occasions. We measured the neural response to rewarding (sight and/or flavor of chocolate) and aversive stimuli (picture of moldy strawberries and/or a less pleasant strawberry taste) using functional magnetic resonance imaging. Volunteers rated pleasantness, intensity, and wanting for each stimulus.

Results:

There were no significant differences between groups in subjective ratings. However, tetrahydrocannabivarin increased responses to chocolate stimuli in the midbrain, anterior cingulate cortex, caudate, and putamen. Tetrahydrocannabivarin also increased responses to aversive stimuli in the amygdala, insula, mid orbitofrontal cortex, caudate, and putamen.

Conclusions:

Our findings are the first to show that treatment with the CB1 neutral antagonist tetrahydrocannabivarin increases neural responding to rewarding and aversive stimuli. This effect profile suggests therapeutic activity in obesity, perhaps with a lowered risk of depressive side effects.     

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

BACKGROUND AND PURPOSE

A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF₁) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan.

EXPERIMENTAL APPROACH

A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF₁ antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF₁ receptor binding of novel compounds can be predicted on the basis of in vitro assays.

KEY RESULTS

The non-competitive antagonist behaviour appeared to be correlated to the CRF₁ receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations.

CONCLUSIONS AND IMPLICATIONS

This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF₁ receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF₁ receptor antagonists in an efficient manner in a drug discovery setting.     

Dopaminergic mechanisms of reinstatement of MDMA-seeking behaviour in rats

BACKGROUND AND PURPOSE

Animal models of drug-seeking suggest that exposure to cues associated with self-administered drugs and drug primes might precipitate relapse via activation of central dopaminergic substrates.

EXPERIMENTAL APPROACH

The effects of priming injections of dopamine and 5-HT agonists on drug-seeking and effects of dopamine antagonists on methylenedioxymethamphetamine (MDMA)-produced potentiation of drug-seeking following extinguished MDMA self-administration were examined.

KEY RESULTS

Drug-seeking was produced by exposure to a light stimulus that had been paired with self-administered MDMA infusions and this effect was potentiated by experimenter-administered injections of the dopamine D₂-like receptor agonist, quinpirole, the indirect agonist, amphetamine and the uptake inhibitor, GBR 12909. Drug-seeking was not elicited by the dopamine D₁-like receptor agonist, SKF 81297 or the non-selective agonist, apomorphine. The 5-HT receptor agonists DOI or mCPP also failed to elicit drug-seeking. The 5-HT uptake inhibitor, clomipramine, attenuated drug-seeking produced by the MDMA-associated stimulus but failed to alter the potentiated response produced by GBR 12909. The D₁ receptor antagonist, SCH 23390 or the D₂ receptor antagonist, eticlopride attenuated the potentiation of drug-seeking produced by MDMA.

CONCLUSIONS AND IMPLICATIONS

These data provide evidence of dopaminergic mechanisms in drug-seeking following extinction of MDMA self-administration. Because tissue levels of 5-HT were significantly decreased following MDMA self-administration, we suggest that MDMA begins to preferentially activate dopaminergic substrates to potentiate the drug-seeking response.     

Angiotensin receptor type 1 antagonists protect against neuronal injury induced by oxygen�Cglucose depletion

BACKGROUND AND PURPOSE

Several clinical trials and in vivo animal experiments have suggested that blockade of angiotensin receptor type 1 (AT₁) improves ischaemic outcomes. However, the mechanism(s) underlying these effects has not been elucidated. Here, we have investigated the protective effects of pretreatment with AT₁ receptor antagonists, losartan or telmisartan, against ischaemic insult to neurons in vitro.

EXPERIMENTAL APPROACH

Primary rat neuron–astrocyte co-cultures and astrocyte-defined medium (ADM)-cultured pure astrocyte cultures were prepared. Ischaemic injury was modelled by oxygen–glucose depletion (OGD) and lactate dehydrogenase release after OGD was measured with or without AT₁ receptor antagonists or agonists (L162313), AT₂ receptor antagonist (PD123319) or agonist (CGP-42112A) pretreatment, for 48 h. Activity of glutamate transporter 1 (GLT-1) was evaluated by [³H]-glutamate uptake assays, after AT₁ receptor agonists or antagonists. Immunoblot and real-time PCR were used for analysis of protein and mRNA levels of GLT-1.

KEY RESULTS

AT₁ receptor agonists augmented OGD-induced cellular damage, which was attenuated by AT₁ receptor antagonists. AT₁ receptor antagonists also suppressed OGD-induced extracellular glutamate release, reactive oxygen species production and nitric oxide generation. GLT-1 expression and glutamate uptake activity were significantly enhanced by AT₁ receptor antagonists and impaired by AT₁ receptor agonists. AT₁ receptor stimulation suppressed both ADM-induced GLT-1 protein expression and mRNA levels. AT₁b receptor knock-down with siRNA enhanced GLT-1 expression. In postnatal (P1–P21) rat brains, protein levels of GLT-1 and AT₁ receptors were inversely correlated.

CONCLUSIONS AND IMPLICATIONS

Suppression of AT₁ receptor stimulation induced GLT-1 up-regulation, which ameliorated effects of ischaemic injury.