Chronic or high dose acute caffeine treatment protects mice against oleic acid-induced acute lung injury via an adenosine A2A receptor-independent mechanism

The antagonism or genetic deletion of adenosine A_2A receptors has been shown to exacerbate tissue damage in acute lung injury. Caffeine, a widely consumed behavioral drug, acts as a non-selective antagonist of A_2A receptor and also has additional pharmacological effects. Thus, the protective vs. deleterious effects of caffeine in acute lung injury should be evaluated. In a murine oleic acid-induced model of acute lung injury, we found that chronic caffeine treatment by drinking water (0.1 g/l or 0.25 g/l for 2 weeks before acute lung injury) or acute caffeine treatment at high dose (i.p. 50 mg/kg, injection, 30 min before acute lung injury) significantly attenuated the lung edema, hemorrhage, neutrophil recruitment as well as the inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) expressions in both of the wild type (WT) and A_2A receptor knockout (KO) mice. This profile was accompanied by increased cAMP levels and up-regulation of A2B receptor mRNAs in the lungs. In contrast, acute caffeine treatment at low dose (i.p. 5 mg/kg or 15 mg/kg, injection, 30 min before acute lung injury) enhanced the inflammation and lung damage in WT mice with decreasing cAMP but not in A_2A receptor KO mice. These results indicate that caffeine either enhances lung damage by antagonizing A_2A receptor or exerts protection against lung damage via A_2A receptor-independent mechanisms, depending on the timing of exposure (chronic vs. acute) and dose of administration (low vs. high). These findings provide new insight of caffeine in acute lung injury and highlight the potential benefit and strategy of caffeine intake or administration for preventing acute lung injury.     

Adenosine A<Subscript>2A</Subscript> receptor deficient mice are partially resistant to limbic seizures

The neuromodulator adenosine, acting through activation of four defined metabotropic receptors called A₁, A_2A, A_2B and A_3, has been proposed as an endogenous anticonvulsant. Here, the consequences of deleting the adenosine A_2A receptor have been examined in different experimental models of epilepsy. A_2AR KO mice were not protected against seizures originating from brainstem structures, namely electroshock-induced seizures. The intensities of seizures induced by pentylenetetrazol or pilocarpine, as well as the percentages of convulsing mice, were significantly reduced in A_2A receptor knockout (A_2AR KO) animals. A_2AR KO mice exhibited reduced pentylenetetrazol-induced kindled seizures, demonstrating an important role of the A_2A receptor in the acquisition of kindling. These data suggest that adenosine stimulating A_2A receptors modulates excitatory neurotransmission and exacerbates limbic seizures. It is therefore suggested that adenosine A_2A receptor antagonists might offer protection from some epileptic syndromes.     

Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

Background and Purpose

Thermostabilization by mutagenesis is one method which has facilitated the determination of high-resolution structures of the adenosine A_2A receptor (A_2AR). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand-binding profiles of these mutants are known, the effects these mutations have on receptor activation and downstream signalling are less well characterized.

Experimental Approach

Here we have investigated the effects of the thermostabilizing mutations on receptor activation using a yeast cell growth assay. The assay employs an engineered Saccharomyces cerevisiae, MMY24, which couples receptor activation to cell growth.

Key Results

Analysis of the receptor activation profile revealed that the wild-type (WT) A_2AR had considerable constitutive activity. In contrast, the Rag23, Rant5 and Rant21 thermostabilized mutants all exhibited no constitutive activity. While the preferentially antagonist-binding mutants Rant5 and Rant21 showed a complete lack of agonist-induced activity, the Rag23 mutant showed high levels of agonist-induced receptor activity. Further analysis using a mutant intermediate between Rag23 and WT indicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reduced G-protein coupling.

Conclusions and Implications

The loss of constitutive activity may be an important feature of these thermostabilized GPCRs. In addition, the constitutively active and agonist-induced active conformations of the A_2AR are distinct.     

Clozapine Reverses Phencyclidine-Induced Desynchronization of Prefrontal Cortex through a 5-HT1A Receptor-Dependent Mechanism

The non-competitive NMDA receptor (NMDA-R) antagonist phencyclidine (PCP)—used as a pharmacological model of schizophrenia—disrupts prefrontal cortex (PFC) activity. PCP markedly increased the discharge rate of pyramidal neurons and reduced slow cortical oscillations (SCO; 0.15–4 Hz) in rat PFC. Both effects were reversed by classical (haloperidol) and atypical (clozapine) antipsychotic drugs. Here we extended these observations to mice brain and examined the potential involvement of 5-HT_2A and 5-HT_1A receptors (5-HT_2AR and 5-HT_1AR, respectively) in the reversal by clozapine of PCP actions. Clozapine shows high in vitro affinity for 5-HT_2AR and behaves as partial agonist in vivo at 5-HT_1AR. We used wild-type (WT) mice and 5-HT_1AR and 5-HT_2AR knockout mice of the same background (C57BL/6) (KO-1A and KO-2A, respectively). Local field potentials (LFPs) were recorded in the PFC of WT, KO-1A, and KO-2A mice. PCP (10 mg/kg, intraperitoneally) reduced SCO equally in WT, KO-2A, and KO-1A mice (58±4%, 42±7%, and 63±7% of pre-drug values, n=23, 13, 11, respectively; p<0.0003). Clozapine (0.5 mg/kg, intraperitoneally) significantly reversed PCP effect in WT and KO-2A mice, but not in KO-1A mice nor in WT mice pretreated with the selective 5-HT_1AR antagonist WAY-100635.The PCP-induced disorganization of PFC activity does not appear to depend on serotonergic function. However, the lack of effect of clozapine in KO-1A mice and the prevention by WAY-100635 indicates that its therapeutic action involves 5-HT_1AR activation without the need to block 5-HT_2AR, as observed with clozapine-induced cortical dopamine release.     

Reduced appetite for caffeine in adenosine A(2A) receptor knockout mice

Adenosine A(2A) receptor knockout mice (A(2A)R KO) were compared to wild-type controls (A(2A)R WT) in a caffeine intake paradigm. When mice had ad libitum access to caffeine (0.3 g/l) and water in a two-bottle paradigm for 12 consecutive days, adenosine A(2A)R KO mice drank less caffeinated solution, demonstrating a reduced appetite for caffeine as compared to adenosine A(2A)R WT mice. These data reveal an important role for the adenosine A(2A) receptor in the appetitive properties of caffeine.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

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.     

Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

Background:

The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons.

Methods:

Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A_2A receptor modulation on intrinsic excitability.

Results:

Activation of adenosine A_2A receptors with the selective A_2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A_2A receptor-mediated effects were blocked by preapplication of a selective A_2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A_2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A_2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons.

Conclusions:

Collectively, these data suggest that adenosine, via activation of A_2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A₁ receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells.     

Clozapine-Induced Locomotor Suppression is Mediated by 5-HT2A Receptors in the Forebrain

The need for safer, more effective therapeutics for the treatment of schizophrenia is widely acknowledged. To optimally target novel pharmacotherapies, in addition to establishing the mechanisms responsible for the beneficial effects of antipsychotics, the pathways underlying the most severe side effects must also be elucidated. Here we investigate the role of serotonin 2A (5-HT_2A), serotonin 2C (5-HT_2C), and dopamine 2 receptors (D₂) in mediating adverse effects associated with canonical first- and second-generation antipsychotic drugs in mice. Wild-type (WT) and 5-HT_2A knockout (KO) mice treated with haloperidol, clozapine, and risperidone were assessed for locomotor activity and catalepsy. WT mice showed a marked reduction in locomotor activity following acute administration of haloperidol and high-dose risperidone, which was most likely secondary to the severe catalepsy caused by these compounds. Clozapine also dramatically reduced locomotor activity, but in the absence of catalepsy. Interestingly, 5-HT_2A KO mice were cataleptic following haloperidol and risperidone, but did not respond to clozapine''s locomotor-suppressing effects. Restoration of 5-HT_2A expression to cortical glutamatergic neurons re-instated the locomotor-suppressing effects of clozapine in the open field. In sum, we confirm that haloperidol and risperidone caused catalepsy in rodents, driven by strong antagonism of D₂. We also demonstrate that clozapine decreases locomotor activity in a 5-HT_2A-dependent manner, in the absence of catalepsy. Moreover, we show that it is the cortical population of 5-HT_2A that mediate the locomotor-suppressing effects of clozapine.     

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.     

Adenosine receptor subtype-selective antagonists in inflammation and hyperalgesia

In this study, we examined the effects of systemic and local administration of the subtype-selective adenosine receptor antagonists PSB-36, PSB-1115, MSX-3, and PSB-10 on inflammation and inflammatory hyperalgesia. Pharmacological blockade of adenosine receptor subtypes after systemic application of antagonists generally led to a decreased edema formation after formalin injection and, with the exception of A₃ receptor antagonism, also after the carrageenan injection. The selective A_2B receptor antagonist PSB-1115 showed a biphasic, dose-dependent effect in the carrageenan test, increasing edema formation at lower doses and reducing it at a high dose. A₁ and A_2B antagonists diminished pain-related behaviors in the first phase of the formalin test, while the second, inflammatory phase was attenuated by A_2B and A₃ antagonists. The A_2B antagonist was particularly potent in reducing inflammatory pain dose-dependently reaching the maximum effect at a low dose of 3 mg/kg. Inflammatory hyperalgesia was totally eliminated by the A_2A antagonist MSX-3 at a dose of 10 mg/kg. In contrast to the A₁ antagonist, the selective antagonists of A_2A, A_2B, and A₃ receptors were also active upon local administration. Our results demonstrate that the blockade of adenosine receptor subtypes can decrease the magnitude of inflammatory responses. Selective A_2A antagonists may be useful for the treatment of inflammatory hyperalgesia, while A_2B antagonists have potential as analgesic drugs for the treatment of inflammatory pain.     

Roles of purines in synaptic modulation evoked by hypercapnia in isolated spinal cord of neonatal rat in vitro

Background and purpose

The purine compounds, adenosine 5′-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord.

Experimental approach

Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods.

Key results

Hypercapnia (20% CO₂) depressed the reflex potentials, which were partially reversed by an adenosine A₁ receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A₁ receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X₇ receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(β-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia.

Conclusions and implications:

These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A₁ receptors.     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x     

Increased brain monoaminergic tone after the NMDA receptor GluN2A subunit gene knockout is responsible for resistance to the hypnotic effect of nitrous oxide

N-methyl-d-aspartate (NMDA) receptors can be inhibited by inhalational anesthetics in vitro at clinically relevant concentrations. Here, to clarify the role of NMDA receptors in anesthetic-induced unconsciousness, we examined the hypnotic properties of isoflurane, sevoflurane and nitrous oxide in NMDA receptor GluN2A subunit knockout mice. The hypnotic properties of inhalational anesthetics were evaluated in mice in the loss of righting reflex (LORR) assay by measuring the 50% concentration for LORR (LORR ED₅₀). Knockout mice displayed isoflurane and sevoflurane LORR ED₅₀ values similar to wild-type controls, indicating no significant contribution of these receptors to the hypnotic action of halogenated anesthetics. However, compared with wild-type controls, mutant mice displayed larger isoflurane LORR ED₅₀ values in the presence of nitrous oxide, indicating a resistance to this gaseous anesthetic. Knockout mice have enhanced brain monoaminergic activity which occurs secondary to NMDA receptor dysfunction, and the observed resistance to the isoflurane LORR ED₅₀-sparing effect of nitrous oxide could be abolished by pretreatment with the dopamine D₂ receptor antagonist droperidol or with the serotonin 5-HT_2A receptor antagonist ketanserin. Thus, resistance to nitrous oxide in knockout mice appears to be a secondary phenomenon of monoaminergic origin and not a direct result of impaired NMDA receptor function. Our results indicate that NMDA receptors are not critically involved in the hypnotic action of conventionally-used inhalational anesthetics. Also, they suggest that increased brain monoaminergic tone can diminish the effects of general anesthesia. Finally, they provide further evidence that changes secondary to genetic manipulation can explain the results obtained in global knockouts.     

Reduced hypophagic effects of <Emphasis Type="Italic">d</Emphasis>-fenfluramine and the 5-HT<Subscript>2C</Subscript> receptor agonist <Emphasis Type="Italic">m</Emphasis>CPP in 5-HT<Subscript>1B</Subscript> receptor knockout mice

Rationale The possible role of compensatory changes in 5-HT_2C receptors in the reduced hypophagic action of d-fenfluramine in 5-HT_1B knockout (KO) mice was assessed by comparing their response to d-fenfluramine and the 5-HT_2C receptor agonist mCPP. In addition we measured 5-HT_2C/A receptor binding in 5-HT_1B KO and wild-type (WT) mice and examined the effects of 5-HT_1B receptor antagonists on d-fenfluramine-induced hypophagia in WT mice.Methods Hypophagic responses to d-fenfluramine (1–30 mg/kg) and mCPP (1–5.6 mg/kg) were measured using a behavioural satiety sequence paradigm. The effects of the 5-HT_1B receptor antagonists GR 127,935 and SB 224289 in opposing the hypophagic action of d-fenfluramine were evaluated in WT mice. The binding of [³H]-mesulergine was compared in the brains of both mouse strains.Results The hypophagic effects of moderate doses of d-fenfluramine and mCPP were attenuated in 5-HT_1B KO mice. Pretreatment of WT mice with the 5-HT_1B/1D receptor antagonist GR 127,935, or food-deprived WT mice with the 5-HT_1B receptor antagonist SB 224289, did not reproduce the reduction in sensitivity to the effects of d-fenfluramine on feeding behaviour observed in 5-HT_1B KO mice. Estimates of 5-HT_2C receptor binding were similar in 5-HT_1B KO and WT mice.Conclusions The hypophagic effect of d-fenfluramine in mice is unlikely to be mediated by the 5-HT_1B receptor. Instead, the evidence suggests that an adaptive change in 5-HT_2C receptor function occurs in 5-HT_1B receptor KO mice and contributes to their reduced response to d-fenfluramine.     

Striatal A2 receptor regulates apomorphine-induced turning in rats with unilateral dopamine denervation

The effect of the purine agonist N-ethylcarboxamido-adenosine (NECA) on apomorphine-induced rotation was investigated in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Intrastriatal administration of NECA on the denervated side caused a dose-dependent inhibition of contralateral rotation. This inhibition was prevented by prior intrastriatal injection of theophylline. The adenosine A₁ receptor antagonist 8-cyclopentyltheophylline was ineffective at concentrations which block this receptor, but effective in preventing the action of NECA at concentrations which block the adenosine A₂ receptor. In the absence of apomorphine, NECA had no effect on behaviour. It is concluded that A₂ receptor activation counteracts apomorphine effects in the striatum. Since the A₂ receptor may be localized to striatal cholinergic neurones, the possible role of these neurones in purine-induced behaviours is discussed.     

Paradoxical constitutive behavioral sensitization to amphetamine in mice lacking 5-HT2A receptors

Rationale

Although locomotor response to d-amphetamine is considered as mediated by an increased release of dopamine in the ventral striatum, blockade of either α1b-adrenergic or 5-HT_2A receptors almost completely inhibits d-amphetamine-induced locomotor response in mice. In agreement with this finding, mice lacking α1b-adrenergic receptors hardly respond to d-amphetamine. However, we show here that, paradoxically, mice lacking 5-HT_2A receptors (5-HT_2A-R KO) exhibit a twofold higher locomotor response to d-amphetamine than wild-type (WT) littermates.

Objectives

To explore why there is a discrepancy between pharmacological and genetic 5-HT_2A receptor blockade.

Materials and methods

Locomotor response and behavioral sensitization to d-amphetamine were measured in presence of prazosin and/or SR46349B, α1b-adrenergic, and 5-HT_2A receptor antagonists, respectively.

Results

Repeating amphetamine injections still increases 5-HT_2A-R KO mice locomotor response to d-amphetamine at a level similar to that of sensitized WT mice. SR46349B (1 mg/kg) has, as expected, no effect in 5-HT_2A-R KO mice. One milligrams per kilogram of prazosin completely blocks d-amphetamine-induced locomotor response in 5-HT_2A-R KO naïve animals but 3 mg/kg is necessary in sensitized 5-HT_2A-R KO mice.

Conclusions

Because naïve 5-HT_2A-R KO mice exhibit an increased cortical noradrenergic response to d-amphetamine, our data suggest that repeated d-amphetamine modifies noradrenergic transmission in 5-HT_2A-R KO mice. Stimulation of specific 5-HT_2A receptors would inhibit noradrenergic neurons. Dramatic decrease in SR46349B efficiency in sensitized WT mice indicates that a disruption of the regulating role of 5-HT_2A receptors on noradrenergic transmission occurs during sensitization and thus represents the physiological basis of behavioral sensitization to d-amphetamine.     

Discovery of Pyridone-Substituted Triazolopyrimidine Dual A2A/A1 AR Antagonists for the Treatment of Ischemic Stroke

Ischemic stroke is a complex systemic disease characterized by high morbidity, disability, and mortality. The activation of the presynaptic adenosine A_2A and A₁ receptors modifies a variety of brain insults from excitotoxicity to stroke. Therefore, the discovery of dual A_2A/A₁ adenosine receptor (AR)-targeting therapeutic compounds could be a strategy for the treatment of ischemic stroke. Inspired by two clinical phase III drugs, ASP-5854 (dual A_2A/A₁ AR antagonist) and preladenant (selective A_2A AR antagonist), and using the hybrid medicinal strategy, we characterized novel pyridone-substituted triazolopyrimidine scaffolds as dual A_2A/A₁ AR antagonists. Among them, compound 1a exerted excellent A_2A/A₁ AR binding affinity (K_i = 5.58/24.2 nM), an antagonistic effect (IC₅₀ = 5.72/25.9 nM), and good metabolic stability in human liver microsomes, rat liver microsomes, and dog liver microsomes. Importantly, compound 1a demonstrated a dose–effect relationship in the oxygen-glucose deprivation/reperfusion (OGD/R)-treated HT22 cell model. These findings support the development of dual A_2A/A₁ AR antagonists as a potential treatment for ischemic stroke.     

Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

Background and Purpose

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats.

Experimental Approach

PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg⁻¹) and 2 weeks later, oral LASSBio-1359 (50 mg·kg⁻¹) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A₁, A_2A, A₃) and of docking with A_2A receptors were also performed.

Key Results

MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A_2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A_2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A_2A receptor agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, were very similar.

Conclusion and Implications

In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A_2A receptors.     

Involvement of the amygdala in the memory-enhancing effects of clenbuterol

CGS 15943A is the first reported nonxanthine adenosine antagonist and it shows high affinity towards A₁ and A₂ receptors. The present data show that CGS 15943A increased in a dose-dependent manner locomotor activity of mice confronted with a free exploratory test without markedly modifying rears or, at low or medium doses, novelty seeking responses. In the light/dark choice procedure, which is especially appropriate for revealing anxiolytic and anxiogenic drug-effects, CGS 15943A decreased the time spent by mice in the lit box and increased the number of transitions. By contrast, the highly selective adenosine A₁ receptor, DPCPX, did not significantly modify the behavior of mice except at high doses, which decreased it in the free exploratory test. It is suggested that the present findings confirm the hypothesis that the behavioral effects of adenosine antagonists are linked to their actions at adenosine A₂ receptors.