SCH 58261: A selective A2A adenosine receptor antagonists

The recent discovery of selective antagonists for the A_2A adenosine receptors has been of great help to further research in this field. One compound, SCH 58261, 5- amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, is playing a key part in a variety of studies. This review describes its pharmacological characteristics as they are emerging in various laboratories. The compound has an affinity in the low nM range (Ki value of 1–2 nM) for A_2A receptors located on membranes from a variety of tissues and cell types, including rat and bovine brain striatum, human platelets, lymphocytes and neutrophils, porcine coronary arteries, and CHO cells transfected with the cloned human A_2A receptors. SCH 58261 has little or no affinity up to the μM range for adenosine A_2B, A₃, or other G protein-coupled receptors. Selectivity for A_2A vs. A₁ receptors varies from 53- to 750-fold, depending on membranes or type of assay. SCH 58261 blocks A_2A-receptor mediated increase of cyclic AMP formation with high potency (e.g., IC₅₀ values between 15 and 20 nM in human white blood cells). The tritiated form of SCH 58261 specifically labels A_2A receptors in discrete regions of the rat brain such as caudate-putamen, nucleus accumbens, and tuberculum olfactorium. In classic in vitro bioassays, such as A_2A-receptor-mediated vasodilation in coronary arteries, SCH 58261 displays competitive antagonistic properties (e.g., pA₂ value of 9.5 in porcine coronary arteries). In assays involving responses mediated by A₁ or A_2B receptors, SCH 58261 shows little or no activity up to concentrations about 100-fold higher than those affecting A_2A receptors (higher concentrations not being testable due to its poor water solubility). In the rat, SCH 58261 enhances locomotor activity (at 3.7 mg/kg ip), increases wakefulness (10 mg/kg ip) and slightly increases both blood pressure and heart rate (10 mg/kg ip). These activities appear to be specifically mediated by A_2A receptors since the drug counteracts the effect of A_2A receptor agonists in some experimental paradigms. In models mimicking CNS disorders, SCH 58261 potentiates the activity of L-DOPA or dopamine receptor agonists in the 6-hydroxydopamine-lesioned rat model. Moreover, the compound reduces brain infarct size in a rat model of cerebral ischemia. Altogether, SCH 58261 and its radiolabeled form have emerged as interesting tools for better understanding the function of A_2A receptors in physiological or altered conditions. Moreover, the neuroprotective properties of SCH 58261 indicate that drugs of this class have a potential for treatment of brain damage produced by Parkinson's disease or stroke. Drug Dev. Res. 42:63–70, 1997. © 1997 Wiley-Liss, Inc.     

Adenosine A1 receptors modulate anxiety in CD1 mice

The effect of the selective adenosine A₁ receptor agonist 2-chloro-N ⁶-cyclopentyladenosine (CCPA) was investigated in CD1 mice by the elevated plus-maze and the light/dark test, two models for measuring anxiety in rodents. CCPA, administered IP, had an anxiolytic effect at 0.3 nmol/kg in the elevated plus-maze and at 1 nmol/kg in the light/dark test. Brain levels of 22 nM were found after administration of 100 nmol/kg CCPA, as measured by ex vivo binding experiments. These values are consistent with the occupancy of adenosine A₁ but not A₂ receptors by CCPA, and suggest that the anxiolytic-like action of CCPA may be mediated by centrally located adenosine A₁ receptors. Both CPT, a selective adenosine A₁ receptor antagonist, and IBMX, a non-selective adenosine antagonist, had an anxiogenic effect in the two tests. It is thus possible that purinergic neurons may be involved in the tonic modulation of affective state in mice. Received: 4 March 1997/Final version: 4 October 1997     

Potent antagonists for the human adenosine A2B receptor. Derivatives of the triazolotriazine adenosine receptor antagonist ZM241385 with high affinity

A series of novel and known 5‐substituted 7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[1,5‐a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist‐induced cyclic AMP response at human A_2B receptors. The known potent adenosine A_2A receptor antagonist, 7‐amino‐2‐(2‐furyl)‐5‐[2‐(4‐hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5‐a][1,3,5]triazine (ZM241385, K_iA_2A = 1.78 nM) had a K_i value of 16.5 nM at A_2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A_2B receptors. A pA₂ value of 7.9 was measured for the inhibition of the cyclic AMP response by A_2B receptors induced by 5′‐N‐ethylcarboxamidoadenosine (NECA). In a series of 5‐phenyl(alkyl)amino analogs the 5‐(2‐phenylethyl)amino analog LUF5452 and the 5‐benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A_2B receptors. Moreover, K_i values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A_2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A_2A receptor (K_i value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A_2B receptor selectivity was achieved. The 5‐(2‐phenylhydrazino) derivative LUF5475 showed a high A_2B receptor affinity (K_i = 7.6 nM), while it was equally active at A_2A receptors, being A_2B receptor‐selective with respect to A₁ and A₃ receptors. Drug Dev. Res. 48:95–103, 1999. © 1999 Wiley‐Liss, Inc.     

Regional differences in the adenosine A(2) receptor-mediated modulation of contractions in rat vas deferens

Adenosine receptors involved in modulation of contractions were characterized in the bisected rat vas deferens by combining pharmacological and immunohistochemical approaches. In both portions, noradrenaline-elicited contractions were enhanced by the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA), and inhibited by the non-selective adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) in the presence of the adenosine A₁ receptor antagonist 1,3-dipropyl-8-cyclopentyl-l,3-dipropylxanthine (DPCPX). The adenosine A_2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamidoadenosine (CGS 21680) also inhibited noradrenaline-elicited contractions but only in the prostatic portion. Contractions elicited by the stable ATP analogue ,β-methyleneATP (,β-MeATP) were inhibited only by NECA in the presence of DPCPX and only in the prostatic portion. This study provides functional evidence for the presence, in both portions of the rat vas deferens, of an adenosine A₁ receptor-mediated enhancement and of an adenosine A₂ receptor-mediated inhibition of contractions. The latter effect is mediated by both A_2A and A_2B subtypes in the prostatic portion but only by the A_2B subtype in the epididymal portion. This regional variation is supported by the immunohistochemical results that revealed an adenosine A_2A receptor immunoreactivity not co-localized with nerve fibres more abundant in the prostatic than in the epididymal portion.     

Adenosine A2A receptor stimulation decreases GAT-1-mediated GABA uptake in the globus pallidus of the rat

We examined modulation of [(3)H]GABA uptake in slices of the rat globus pallidus because stimulation of adenosine A(2A) receptors increases extracellular GABA in this structure. Pharmacological analysis showed that GAT-1 is the main transporter present in these slices. Both adenosine and the A(2A) agonist CGS 21680 reduced GABA uptake. Antagonist ZM 241385 prevented these effects. Agents that increase protein kinase A activity like forskolin and 8-bromo-cAMP also inhibited GABA uptake. The inhibition of uptake produced by these substances and by CGS 21680 was prevented by the protein kinase A blocker H-89. The protein phosphatase blocker okadaic acid reduced uptake; this effect and the response to CGS 21680 were not additive. The effective concentrations of adenosine (EC(50)=15.2microM) are within the range measured in the interstitial fluid under some physiological conditions. Thus, inhibition of uptake may be important in increasing interstitial GABA during endogenous adenosine release.     

Actions of adenosine A2A receptor antagonist KW-6002 on drug-induced catalepsy and hypokinesia caused by reserpine or MPTP

Rationale: Current treatment of Parkinson’s disease (PD) is based on dopamine replacement therapy, but this leads to long term complications, including dyskinesia. Adenosine A_2A receptors are particularly abundant in the striatum and would be a target for an alternative approach to the treatment of PD. Objectives: The purpose of this study is to examine the efficacy and potency of the novel selective adenosine A_2A receptor antagonist (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dhydro-1H-purine-2,6-dione (KW-6002) in ameliorating the motor deficits in various mouse models of Parkinson’s disease. Methods: We evaluated the efficacy and potency of KW-6002 and other reference compounds in the selective adenosine A_2A receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamidoadenosine (CGS 21680)-, haloperidol- or reserpine-induced catalepsy models. The effect of KW-6002 on reserpine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride(MPTP)-induced hypolocomotion was also examined. Results: The ED₅₀s of KW-6002 in the reversal of CGS21680-induced and reserpine-induced catalepsy were 0.05 mg/kg, PO and 0.26 mg/kg, PO, respectively. Compared to the ED₅₀ of other adenosine antagonists and dopamine agonist drugs, KW-6002 is over 10 times as potent in these models. KW-6002 also ameliorated the hypolocomotion (minimum effective dose; 0.16 mg/kg) induced by nigral dopaminergic dysfunction with MPTP or reserpine treatment. Combined administrations of subthreshold doses of KW-6002 and l-dopa (50 mg/kg, PO) exerted prominent effects on haloperidol-induced and reserpine-induced catalepsy, suggesting that there may be a synergism between the adenosine A_2A receptor antagonist KW-6002 and dopaminergic agents. Conclusions: To our knowledge, KW-6002 is the most potent and orally active adenosine A_2A receptor antagonist in experimental models of Parkinson’s disease, and may offer a new therapeutic approach to the treatment of Parkinson’s disease. Received: 22 March 1999 / Final version: 25 May 1999     

G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([³H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins. Received: 22 July 1998 / Accepted: 18 January 1999     

Effects of CGS 21680, a selective adenosine A2A receptor agonist, on allergic airways inflammation in the rat

We have investigated the effect of 2(4-((2-carboxymethyl)phenyl)ethylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680), a potent and selective agonist at adenosine A2A receptors, on pulmonary inflammation induced by allergen challenge in the ovalbumin-sensitised, Brown Norway rat. Aerosol administration of ovalbumin (5 mg x ml(-1) for 60 min; calculated dose 0.4 mg x kg(-1)) induced increases in bronchoalveolar lavage fluid leukocyte numbers, protein content and myeloperoxidase and eosinophil peroxidase activities measured 24 h post challenge. CGS 21680 (10 and 100 microg x kg(-1) given intratracheally (i.t.) 30 min before and 3 h after allergen challenge) inhibited dose-dependently all the parameters of inflammation. Qualitatively similar results were obtained with the glucocorticosteroid, budesonide (0.1, 1 and 10 mg x kg(-1) given 3 h prior to ovalbumin challenge). CGS 21680 given i.t. reduced blood pressure in anaesthetised rats at similar doses to those at which anti-inflammatory effects were manifested. Both the anti-inflammatory and hypotensive responses to CGS 21680 were blocked by pretreatment with the selective adenosine A2A receptor antagonist, 4-(2-(7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a(1,3,5)triazin-5-yl amino)ethyl)phenol (ZM 241385), 3 mg x kg(-1) p.o., 1 h prior to the agonist. Thus, CGS 21680 manifests broad-spectrum anti-inflammatory activity in a model of allergic asthma in the Brown Norway rat through activation of adenosine A2A receptors. The striking similarity to budesonide, a clinically used anti-inflammatory agent, suggests that adenosine A2A receptor agonists may be useful alternatives to glucocorticosteroids in the treatment of asthma.     

Behavioural profiles in the mouse defence test battery suggest anxiolytic potential of 5-HT1A receptor antagonists

  Rationale: Compounds varying in selectivity as 5-HT_1A receptor antagonists have recently been reported to produce anxiolytic-like effects comparable to those of benzodiazepines in the mouse elevated plus-maze procedure. Objective: In view of the potential clinical significance of these findings, the present experiments compared the behavioural effects of diazepam (0.5–3.0 mg/kg) with those of several non-selective 5-HT_1A receptor antagonists [NAN-190, 0.1–3.0 mg/kg, MM-77, 0.03–1.0 mg/kg, (S)-UH-301, 0.3–3.0 mg/kg and pindobind-5-HT_1A, 0.03–1.0 mg/kg], and three selective 5-HT_1A receptor antagonists (WAY100635, 0.01–3.0 mg/kg, p-MPPI, 0.1–3.0 mg/kg and SL88.0338, 0.3–3.0 mg/kg) in the mouse defence test battery (MDTB). Methods: In this well-validated anxiolytic screening test, Swiss mice are directly confronted with a natural threat (a rat) as well as situations associated with this threat. Primary measures taken during and after rat confrontation were flight, risk assessment (RA), defensive threat/attack and escape attempts. Results: Diazepam significantly decreased flight reactions after the rat was introduced into the runway, reduced RA activities of mice chased by the rat, increased RA responses displayed when subjects were constrained in a straight alley and reduced defensive upright postures and biting upon forced contact. All the selective 5-HT_1A receptor antagonists and NAN-190 also reduced flight, RA in the chase test, and defensive threat and attack behaviours. (S)-UH-301 and pindobind-5-HT_1A reduced RA in the chase test, but only partially modified defensive threat and attack. Unlike the other drugs tested, MM-77 produced significant effects only at doses which also markedly reduced spontaneous locomotor activity, suggesting a behaviourally non-specific action. In contrast to diazepam, the 5-HT_1A receptor ligands failed to affect RA in the straight alley test. Following removal of the rat from the test area, only diazepam and (S)-UH-301 reduced escape behaviour (contextual defence) at doses which did not decrease locomotion. Overall, the present findings indicate that except for one RA behaviour and escape responses, the 5-HT_1A receptor ligands studied modified the same defensive behaviours as diazepam, suggesting potential therapeutic efficacy in the management of anxiety disorders. However, the magnitude of the effects of the 5-HT_1A compounds on defence was generally smaller than that of the benzodiazepine. Conclusion: As all of the 5-HT_1A compounds tested in this series share antagonistic activity in models of postsynaptic 5-HT_1A receptor function, it is proposed that this action accounts for their effects on defence. Received: 29 June 1998 / Final version: 16 December 1998     

The adenosine A1 receptor contributes to the stimulatory, but not the inhibitory effect of caffeine on locomotion: a study in mice lacking adenosine A1 and/or A2A receptors

Caffeine has biphasic effects on locomotion, and blockade of the adenosine A(2A) receptor (A2AR) is necessary for the stimulatory effect of low doses of caffeine, but not for the locomotor depressant effect observed at high doses. We wanted to elucidate the role of the adenosine A(1) receptor (A1R) in mediating the locomotor effects of increasing doses of caffeine using wild-type mice (A1R(WT)), mice heterozygous for (A1R(HET)), and mice lacking the adenosine A(1) receptor (A1R(KO)). Caffeine had the typical biphasic dose-effect relationship in all three genotypes, but the stimulatory action of caffeine was facilitated in the A1R(KO) mice. In order to investigate the interaction between blockade of A1Rs and A2ARs, mice lacking both receptors (A1R(KO)/A2AR(KO)) were tested. Regardless of A1R genotype, animals lacking A2AR were not stimulated by caffeine, whereas animals heterozygous for A2AR were. As expected, the A1R is not crucial for the stimulatory effect of caffeine, but seems to modulate the effect of caffeine exerted via A2AR blockade. Furthermore, these results suggest that the inhibitory effect of high doses of caffeine is due neither to blockade of the A1R, nor of the A2AR, and an effect independent of these adenosine receptors is likely.     

The effects of adenosine A2A receptor antagonists on haloperidol-induced movement disorders in primates

RATIONALE: Adenosine and dopamine interact within the striatum to control striatopallidal output and globus pallidus GABA release. Manipulating striatal adenosine transmission via blockade of the A(2A) receptor subtype can compensate for the reduced dopamine activity within the striatum that underlies movement disorders such as antipsychotic-induced extrapyramidal syndrome (EPS) and Parkinson's disease (PD). Preclinical studies in the rat have demonstrated that adenosine A(2A) receptor antagonists can attenuate behaviors reflecting reduced dopamine activity, such as haloperidol-induced catalepsy and hypoactivity. OBJECTIVES: In the present studies using nonhuman primates, adenosine antagonists were tested against haloperidol-induced EPS in Cebus apella and haloperidol-induced catalepsy in Saimiri sciureus (squirrel monkey). Specifically, the A(2A) receptor antagonists, SCH 412348 (0.3-30 mg/kg PO) and KW-6002 (3-100 mg/kg PO); the A(1)/A(2A) receptor antagonist, caffeine (1-30 mg/kg PO and IM); and the A(1) receptor antagonist, DPCPX (3-30 mg/kg PO) were tested in at least one of these models. RESULTS: SCH 412348 (10-30 mg/kg), KW-6002 (57-100 mg/kg), and caffeine (30 mg/kg) significantly increased the time to EPS onset. Additionally, SCH 412348, KW-6002, and caffeine afforded protection from the onset of EPS for at least 6 h in some of the primates. SCH 412348 (10 mg/kg) and caffeine (10 mg/kg) significantly reduced haloperidol-induced catalepsy. DPCPX produced a very slight attenuation of EPS at 30 mg/kg, but had no effect on catalepsy. CONCLUSIONS: These findings suggest that adenosine A(2A) receptor antagonists may represent an effective treatment for the motor impairments associated with both antipsychotic-induced EPS and PD.     

Effects of the selective angiotensin II receptor antagonists losartan and PD123177 in animal models of anxiety and memory

There is increasing interest in the potential functional role of the octapeptide angiotensin II (AII) in psychiatric and cognitive disorders. The novel angiotensin II (AII) receptor antagonists, losartan and PD123177, selective for the AT₁ and AT₂ receptor subtypes respectively, constitute important pharmacological tools for the assessment of the behavioural consequences of modulation of AII function. The present series of studies investigated the effects of each compound in two animal models of anxiety, the rat elevated zero-maze and mouse light/dark box, and two models of working memory in the rat, the operant delayed matching to position (DMTP) task and the spatial reinforced alternation test in the T-maze. Our data indicate that both compounds (0.01–10 mg/kg SC) were without significant effect in any of the behavioural assays. Using the present methods and strains of laboratory rodents, these findings provide no support for the involvement of AII receptor function in the mediation of anxiety or working memory.     

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.     

The role of 5-HT receptor subtypes in the anxiolytic effects of selective serotonin reuptake inhibitors in the rat ultrasonic vocalization test

 We evaluated whether the anxiolytic effects of selective serotonin reuptake inhibitors (SSRIs) in the rat ultrasonic vocalization (USV) test are preferentially mediated by (indirect) activation of 5-HT_1A, 5-HT_1B/1D, 5-HT_2A, 5-HT₃ or 5-HT₄ receptors. The SSRIs, paroxetine (ED₅₀ in mg/kg, IP: 6.9), citalopram (6.5), fluvoxamine (11.7) and fluoxetine (>30), dose dependently reduced shock-induced USV. The effects of paroxetine (3.0 mg/kg, IP) were not blocked by the selective 5-HT_1A receptor antagonist, WAY-100635 (3.0 mg/kg, IP), the 5-HT_1B/1D receptor antagonist, GR 127935 (30 mg/kg, IP), the nonselective 5-HT_2A receptor antagonists, ritanserin (3.0 mg/kg, IP) and ketanserin (1.0 mg/kg, IP), the 5-HT₃ receptor antagonist, ondansetron (0.1 mg/kg, IP), or the 5-HT₄ receptor antagonist, GR 125487D (3.0 mg/kg, SC). In contrast, the selective 5-HT_2A receptor antagonist, MDL 100,907 (0.1 mg/kg, IP), completely prevented the paroxetine-induced reduction of USV. Under similar conditions, WAY-100635 blocked the anxiolytic-like effects of the selective 5-HT_1A receptor agonist, 8-OH-DPAT [(±)-8-hydroxy-2-(di-n-propylamino)tetralin, 1.0 mg/kg, IP], and ritanserin, ketanserin, and MDL 100,907 blocked the anxiolytic-like effects of the mixed 5-HT_2A/2C receptor agonist, DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 3.0 mg/kg, IP]. WAY-100635 (1.0 mg/kg, IP) in combination with ritanserin (3.0 mg/kg, IP), but not ondansetron (0.1 mg/kg, IP), GR 125487D (3.0 mg/ kg, SC), or GR 127935 (30 mg/kg, IP), attenuated the USV reducing effects of paroxetine. Although the results suggest that selective stimulation of 5-HT_1A and 5-HT_2A receptors produces a decrease of USV, we postulate that only 5-HT_2A receptors play a pivotal role in the effects of SSRIs in this model of anxiety. Received: 19 May 1997 / Final version: 21 July 1997     

Mechanism of tolerance development to 2,5-dimethoxy-4-iodoamphetamine in rats: down-regulation of the 5-HT2A, but not 5-HT2C, receptor

  Rationale: Defining the mechanism of tolerance development to hallucinogenic drugs will help to explain their mechanism of action. Objectives: The present study was conducted to determine first, if tolerance develops to the discriminative stimulus (DS) properties of the hallucinogen, 2,5 dimethoxy-4-iodo-amphetamine (DOI) and second, the mechanism mediating tolerance. Methods: Rats were trained to discriminate 0.75 mg/kg DOI from saline on a concurrent VI-30-min schedule of reinforcement with a 15-min time-out for incorrect responses. To evaluate tolerance development, rats were assigned to one of four groups and treated with either chronic saline or chronic DOI. Prior to chronic treatment, two groups were tested for choice behavior following vehicle administration while the remaining two groups were tested following the administration of 0.375 mg/kg DOI. One group from each pre-test condition was injected with either saline or DOI (1 mg/kg) for 8 days. Twenty-four hours after the last chronic injection the pre-test treatments were replicated. Using receptor autoradiography, the density of 5-HT_2A and 5-HT_2C receptors was measured in independent groups of rats that had received identical treatment conditions. Results: Animals receiving chronic DOI showed a 60% decrease in DOI lever responding (from 100% to 40%) when tested on 0.375 mg/kg DOI, while animals receiving chronic saline showed no change in percent choice (100%) on the DOI lever. Significant changes in binding were observed in 5-HT_2A receptors but not 5-HT_2C receptors. The results of tests with antagonists were consistent with the changes in binding. Conclusions: These results suggest that behavioral tolerance to DOI reflects neuroadaptive changes in 5-HT_2A receptors. Received: 17 July 1998 / Final version: 19 January 1999     

Prolactin response to nemonapride, a selective antagonist for D2 like dopamine receptors, in schizophrenic patients in relation to Taq1A polymorphism of DRD2 gene

The dopamine D₂ receptor (DRD2) gene has a Taq1A restriction fragment length polymorphism yielding two alleles, A1 and A2. It has been shown that the subjects with less frequent allele, the A1 allele, have lower density and diminished function of DRD2 in the striatum, compared to those with no A1 allele. In the present study, the relationship between this polymorphism and prolactin response to nemonapride, an antipsychotic drug with selective and potent DRD2 antagonistic property, was investigated in 25 Japanese schizophrenic inpatients (13 males, 12 females). The daily dose of nemonapride was fixed at 18 mg, and the duration of treatment was 3 weeks. Taq1A genotypes were determined by the polymerase chain reaction method. Plasma prolactin concentrations were measured by enzyme immunoassay. The subjects were divided into four subgroups by gender and Taq1A genotypes, i.e., six males and eight females with the A1 allele, seven males and four females with no A1 allele. The Δprolactin (change from the pretreatment concentration) at 1 week was significantly (P<0.05) higher in females with the A1 allele (78.0±47.1 ng/ml) than in males with the A1 allele (33.4±14.0 ng/ml) or with no A1 allele (29.5±24.8 ng/ml). In addition, Δprolactin at 3 weeks was significantly (P<0.05) higher in females with the A1 allele (98.1±67.9 ng/ml) than in females with no A1 allele (33.4±24.6 ng/ml), males with the A1 allele (29.1±17.3 ng/ml) or males with no A1 allele (28.6±22.0 ng/ml). The present study thus suggests that female patients with the A1 allele show a greater prolactin response to nemonapride, who may have a high risk for adverse effects associated with neuroleptic-induced hyperprolactinemia. Received: 27 July 1999 / Final version: 9 December 1999     

Evidence for the involvement of the adenosine A(2A) receptor in the lowered susceptibility to pentylenetetrazol-induced seizures produced in mice by long-term treatment with caffeine

Long-term caffeine intake has been reported to decrease the susceptibility to convulsants in mice. Occurrence of seizures following long-term oral administration of caffeine (0.3g/l) was investigated using adenosine A(2A) receptor knockout (A(2A)R KO) and control (A(2A)R WT) mice. Clonic seizures induced by acute pentylenetetrazol (PTZ, 50mg/kg i.p.) were significantly attenuated in adenosine A(2A)R KO mice drinking only water and reduced by a 14-day caffeine treatment in adenosine A(2A)R WT mice. In addition we showed a protecting effect of a 21-day caffeine treatment in A(2A)R WT mice against kindled seizures induced by PTZ in an increasing dose schedule. Summing up, these protective effects against PTZ-induced seizures occurring when adenosine A(2A)R is absent or chronically blocked by a relevant dose of caffeine may be related to a decreased neuronal excitability.     

Yohimbine produces antinociception in the formalin test in rats: involvement of serotonin1A receptors

Rationale: Previous studies have suggested that the α₂-adrenergic receptor antagonist yohimbine produced antinociceptive effects in the formalin test in rats. However, yohimbine is also an agonist at serotonin (5-HT)_1A receptors, suggesting the possibility that the antinociceptive effects of yohimbine might be mediated via these receptors. Objective: The purpose of the present studies was to evaluate the potential role of 5-HT_1A receptors in mediating the antinociceptive effects of yohimbine. Methods: The antinociceptive effects of yohimbine were evaluated using the formalin test in rats. Results: Yohimbine (2.5–10 mg/kg s.c.) produced dose-related antinociception during both phase I and phase II of the formalin test, and was approximately equipotent and equiefficacious to morphine. The selective 5-HT_1A receptor antagonist WAY 100,635 (0.03–3.0 mg/kg s.c.) produced a partial reversal of yohimbine. In comparison, the selective 5-HT_1A receptor agonist (±)8-hydroxy- dipropylaminotetralin HBr (8OH-DPAT; 1.0 mg/kg s.c.) also produced a dose-related antinociception in the formalin test, although 8OH-DPAT was completely reversed by WAY 100,635 (3.0 mg/kg s.c.). The antinociceptive effects of yohimbine were not antagonized by the 5-HT_1B/1D antagonist GR 127935 (1.0 mg/kg and 3.0 mg/kg s.c.), the 5-HT₂ antagonist LY53857 (1.0 mg/kg s.c.), or the 5-HT₃ antagonist zatosetron (3.0 mg/kg s.c.). Conclusions: The present results demonstrate that yohimbine produces a dose-related antinociception in the formalin test in rats which is mediated in part by the agonistic actions at 5-HT_1A receptors. Received: 10 September 1999 / Final version: 5 November 1999     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

Enhancement of the acoustic startle response in rats by the dopamine D1 receptor agonist SKF 82958

  Rationale: The present series of experiments was conducted in order to assess the nature of dopaminergic modulation of the acoustic startle response using agonists and antagonists specific for dopamine D₁ and D₂ receptors. Objectives: The objective of the present study was to demonstrate an enhancement of the acoustic startle response by dopamine D₁ receptor agonists and to characterize this effect pharmacologically in terms of dose-response and selective antagonism at both the dopamine D₁ and D₂ receptor using a varied range of startle-eliciting intensities. Methods: Male Sprague-Dawley rats were injected subcutaneously with the dopamine D₁ receptor agonist SKF 82958 (0, 0.01, 0.1, 1, or 3 mg/kg) or SKF 81297 (3 mg/kg) and their startle response was measured across a range of startle-eliciting intensities. For testing with the dopamine D₁ or D₂ receptor antagonists, animals received injections of either SCH 23390 (0.01 and 0.1 mg/kg) or raclopride (0.1 and 1 mg/kg) 10 min before the challenge with SKF 82958 (1 mg/kg). Results: Systemic administration of SKF 82958 produced a marked enhancement of startle over a wide range of startle intensities. This effect was dose-dependent, with a dose of 1 mg/kg producing the maximal amount of startle enhancement at each intensity. SKF 81297 (3 mg/kg) also produced a robust enhancement of startle. Pretreatment with SCH 23390 produced a dose-dependent blockade of the enhancement of startle by SKF 82958. Pretreatment with raclopride blocked the enhancement of startle by SKF 82958 at the low intensities and attenuated the enhancement at the high intensities. Conclusions: These data suggest that dopamine D₁ receptor agonists enhance the acoustic startle response. Furthermore, this effect is dependent on a cooperative type of D₁/D₂ receptor interaction whereby D₂ receptor activation is necessary for the full expression of the D₁ receptor-mediated enhancement of startle. Received: 10 October 1998 / Final version: 11 January 1999