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We aimed to evaluate the novel high-affinity and relatively lipophilic CB(1) receptor (CB(1)R) antagonist radioligand [(125)I]SD7015 for SPECT imaging of CB(1)Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CT(PLUS) (Mediso, Budapest, Hungary), in knock-out CB(1) receptor knock-out (CB(1)R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB(1)R-/- mice (n=3) and C57BL6 wildtype mice (n=7) under urethane anaesthesia after injecting [(125)I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4μm-thin cryostat sections of the excised brains. %SUV was 8.6±3.6 (average±SD) in CB(1)R-/- mice and 22.1±12.4 in wildtype mice between 2 and 4h after injection (p

Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²⁵I]epibatidine and [¹²⁵I]α-bungarotoxin binding to α4β2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4β2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4β2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors.

Concentration–response curves (CRCs) of adenosine receptor (AR) agonists, NECA (nonspecific), CCPA (A1 specific), CGS-216870 (A2A specific), BAY 60-6583 (A2B specific), and Cl-IB-MECA (A3 specific) for mesenteric arteries (MAs) from 4 AR knockout (KO) mice (A1, A2A, A2B, and A3) and their wild type (WT) were constructed. The messenger RNA expression of MAs from KO mice and WT were also studied. Adenosine (10−5 to 10−4 M) and NECA (10−6 to 10−5 M) induced relaxation in all mice except A2B KO mice, which only showed constriction by adenosine at 10−6 to 10−4 and NECA at 10−8 to 10−5 M. The CCPA induced a significant constriction at 10−8 and 10−7 M in all mice, except A1KO. BAY 60-6583 induced relaxation (10−7 to 10−5 M) in WT and no response in A2BKO except at 10−5 M. The CRCs for BAY 60-6583 in A1, A2A, and A3 KO mice shifted to the left when compared with WT mice, suggesting an upregulation of A2B AR. No responses were noted to CGS-21680 in all mice. Cl-IB-MECA only induced relaxation at concentration greater than 10−7 M, and no differences were found between different KO mice. The CRC for Bay 60-6583 was not significantly changed in the presence of 10−5 M of L-NAME, 10−6 M of indomethacin, or both. Our data suggest that A2B AR is the predominant AR subtype and the effect may be endothelial independent, whereas A1 AR plays a significant modulatory role in mouse MAs.

Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B. In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((π/4) × D2 × VTI × HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states., SCOPUS: ar.j, info:eu-repo/semantics/published

Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour. © 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.

Knockout mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and this may be due to the presence of pronociceptive A(2A) receptors on sensory nerves. In support of this hypothesis, we have recently shown that in A(2A) receptor knockout mice there are marked reductions in the changes of two markers of spinal cord neuronal activity, [(3)H]MK801 binding to NMDA receptors and uptake of [(14)C]-2-deoxyglucose, in response to formalin injection. We now report that following a more prolonged inflammatory stimulus, consisting of intraplantar injections of PGE(2) and paw pressure, there was in contrast an increase in [(3)H]MK801 binding and [(14)C]-2-deoxyglucose uptake in the spinal cords of the A(2A) receptor knockout mice which was much greater than in the wild-type mice. This increase suggests that when there is a pronounced inflammatory component to the stimulus, loss of inhibitory A(2A) receptors on inflammatory cells outweighs the loss of pronociceptive A(2A) receptors on peripheral nerves so that overall there is an increase in nociceptive signalling. This implies that although A(2A) antagonists have antinociceptive effects they may have only limited use as analgesics in chronic inflammatory pain.

Several techniques exist to study the rate of glucose uptake and metabolism in the brain but most of them are not sufficiently robust to permit extensive pharmacological analysis. Here we optimized an in vitro measurement of the simultaneous accumulation of the metabolizable and non-metabolizable 3H and 14C d-glucose analogues; permitting convenient large-scale studies on glucose uptake and metabolism in brain slices. Next, we performed an extensive pharmacological characterization on the putative glucoregulator role of the endocannabinoid system in the hippocampal slices of the rat, and the wild-type and the CB 1 cannabinoid receptor (CB 1R) knockout mice. We observed that 3H-3-O-methylglucose is a poor substrate to measure glucose uptake in the hippocampus. 3H-2-deoxyglucose is a better substrate but its uptake is still lower than that of 14C-U-d-glucose, from which the slices constantly metabolize and dissipate 14C atoms. Thus, uptake and the metabolism values are not to be used as standalones but as differences between a control and a treatment. The CB 1R knockout mice exhibited ∿10% less glucose uptake and glucose carbon atom dissipation in comparison with the wild-type mice. This may represent congenital defects as acute treatments of the rat and mouse slices with cannabinoid agonists, antagonists and inhibitors of endocannabinoid uptake/metabolism failed to induce robust changes in either the uptake or the metabolism of glucose. In summary, we report here an optimized technique ideal to complement other metabolic approaches of high spatiotemporal resolution. This technique allowed us concluding that CB 1Rs are at least indirectly involved in hippocampal glucoregulation. © 2011 Elsevier B.V.

Ledent C, Parma J, Dumont J, Vassart G, Targovnik H. Molecular genetics of thyroid diseases. Eur J Endocrinol 1994;130:8–14. ISSN 0804–4643 Over the past 20 years, recombinant deoxyribonucleic acid technology has led to the cloning of three major thyroid specific protein genes (thyroglobulin, thyroperoxidase and thyrotrophin receptor), which happen to be also the main thyroid autoantigens implicated in thyroid diseases. In this context, the impact that molecular genetics has had on the understanding of aetiopathogeny and diagnosis of thyroid diseases is summarized, with special emphasis on a recently discovered genetic mechanism responsible for toxic nodules. One fruitful outcome of the basic research on thyroid-specific gene expression has been the possibility of targeting the expression of a series of genes to the thyroid in transgenic mice. The result is the availability of mouse models mimicking human thyroid diseases such as destructive hypothyroidism, hyperactive thyroid adenomas and thyroid cancers exhibiting varying levels of differentiation. Gilbert Vassart, Institut de Recherche Interdisciplinaire, Faculté de médecine, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, 1070 Bruxelles, Belgium

Previously, we have shown that A2A adenosine receptor (A2AAR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A2AAR is still not fully understood. In this study, we hypothesize that, in the absence of A2AAR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A1 receptor (A1AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A2AKO mice were precontracted with submaximal dose of phenylephrine (10−6 M), and concentration responses for selective A1AR, A2AAR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20-HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2- p-(2-Carboxyethyl)-phenethylamino-5′- N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A2AAR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10−5 M) and also with removal of endothelium. A1 agonist, 2-chloro- N6-cyclopentyladenosine (CCPA) produced higher contraction in A2AKO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10−6 M, P < 0.05). 20-HETE produced higher contraction in A2AKO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10−7 M, P < 0.05). Contraction to CCPA in WT and A2AKO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10−6 M), chelerythrine chloride (nonselective PKC blocker; 10−6 M), Gö-6976 (selective PKC-α inhibitor; 10−7 M), and HET0016 (20-HETE inhibitor; 10−5 M). Also, contraction to 20-HETE in WT and A2AKO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A1AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A2AKO compared with WT ( P < 0.05), while expression of Cyp2c29 was significantly higher in WT. CCPA (10−6 M) increased the protein expression of PKC-α and phosphorylated-ERK1/2, while HET0016 significantly reduced the CCPA-induced increase in expression of these proteins. These data suggest that, in the absence of A2AAR, Cyp4a induces vasoconstriction through MAPK via upregulation of A1AR and PKC-α.

The endocannabinoid system is known to play a role in regulating myocardial contractility, but the influence of cannabinoid receptor 1 (CB1) deficiency on chronic heart failure (CHF) remains unclear. In this study we attempted to investigate the effect of CB1 deficiency on CHF induced by pressure overload and the possible mechanisms involved.A CHF model was created by transverse aortic constriction (TAC) in both CB1 knockout mice and wild-type mice. CB1 knockout mice showed a marked increase of mortality due to CHF from 4 to 8 weeks after TAC (p=0.021). Five weeks after TAC, in contrast to wild-type mice, CB1 knockout mice had a higher left ventricular (LV) end-diastolic pressure, lower rate of LV pressure change (± dp/dt max), lower LV contractility index, and a larger heart weight to body weight ratio and lung weight to body weight ratio compared with wild-type mice (all p0.05-0.001). Phosphorylation of the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (P38 and ERK) was higher in CB1 knockout mice than that in wild-type mice. In cultured neonatal rat cardiomyocytes, a CB1 agonist reduced cAMP production stimulated by isoproterenol or forskolin, and suppressed phosphorylation of the EGFR, P38, and ERK, while the inhibitory effect of a CB1 agonist on EGFR phosphorylation was abrogated by CB1 knockdown.These findings indicate that cannabinoid receptor 1 inactivation promotes cardiac remodeling by enhancing the activity of the epidermal growth factor receptor and mitogen-activated protein kinases.

G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D(2) receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D(2) receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D(2) receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D(2) receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D(2) receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D(2) receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D(2) receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine release in other brain areas in response to persistent dopamine release and administration of psychostimulants.

Brain dopamine (DA) and adenosine interact in the regulation of behavioral activation and effort-related processes. In the present studies, a T-maze task was developed in mice for the assessment of effort-related decision making. With this task, the two arms of the maze have different reinforcement densities, and a vertical barrier is positioned in the arm with the higher density (HD), presenting the animal with an effort-related challenge. Under control conditions mice prefer the HD arm, and climb the barrier to obtain the larger amount of food. The DA D2 receptor antagonist haloperidol decreased selection of the HD arm and increased selection of the arm with the low density of reinforcement. However, the HD arm was still the preferred choice in haloperidol-treated mice trained with barriers in both arms. Pre-feeding the mice to reduce food motivation dramatically increased omissions, an effect that was distinct from the actions of haloperidol. Co-administration of theophylline, a nonselective adenosine receptor antagonist, partially reversed the effects of haloperidol. This effect seems to be mediated by the A2A receptor but not the A1 receptor, since the A2A antagonist MSX-3, but not the A1 antagonist CPT, dose dependently reversed the effects of haloperidol on effort-related choice and on c-Fos expression in the dorsal striatum and nucleus accumbens. In addition, adenosine A2A receptor knockout mice were resistant to the effects of haloperidol on effort-related choice in the maze. These results indicate that DA D2 and adenosine A2A receptors interact to regulate effort-related decision making and effort expenditure in mice. This work was supported by a grant to Mercè Correa from Fundació Bancaixa-UJI (P1.1B2010-43), to John D. Salamone from the National Institute of Mental Health (MH078023), to Olga Valverde from MCINN (SAF2010-1593) and ISCIII RTA (RD06/0001/ 1001). Christa E. Müller and Younis Baqi were funded by the BMBF, 01EW0911 in the framework of ERA-NET-NEURON.

Aims We investigated the influence of type one cannabinoid receptor (CB1) deficiency on acute heart failure (AHF) and the underlying mechanism. Acute heart failure syndrome is an important clinical problem because of its high morbidity and mortality rates. Activation of CB1 induces vascular dilation and reinforces the properties of morphine, long-standing therapies for AHF syndrome, but the effect of endogenous CB1 activation on AHF is largely unknown. Methods and results Acute heart failure mouse model characterized by hypertension and pulmonary oedema was created by using transverse aortic constriction (TAC). Mortality, echocardiography, haemodynamic, morphology, and circulatory catecholamine levels in response to TAC were evaluated in CB1 knockout (KO) and wild-type mice. Type one cannabinoid receptor KO mice had a much higher mortality rate at 1 week after TAC attributable to AHF (65 vs. 11%, P < 0.001). One hour after TAC, CB1 KO mice had significant larger lung weight to body weight ratio (LW/BW, 14.53 + 1.09 mg/g in KO vs. 10.42 + 0.36 mg/g in WT, P < 0.01) and higher plasma epinephrine levels (9720 + 1226 pg/mL vs. 6378 + 832 pg/mL, P < 0.05). Pharmacological activation of CB1 reduced LW/BW in wild-type mice. Administration of epinephrine to wild-type TAC mice significantly increased left ventricular end-diastolic pressure and LW/BW, while CB1 agonists reduced the LW/BW and the plasma levels of catecholamine and increased myocardial activity of AMP-activated protein kinase. Conclusion Endogenous activation of CB1 in mice has cardiac protection in AHF, which is attributable to the inhibition of excessive sympathetic activation.

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210CP55,490WIN55,212-22-AGTHC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1-1 µM), 2-AG directly affected the functions of recombinant homomeric alpha1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ~300 milliseconds. Addition of 1 µM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4-10 Hz) application of short (2-ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor-knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10-20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner.

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble >find me> signals, 2) recognition and phagocytosis via cell surface-presenting >eat me> signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor d. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation. Copyright © 2011 by The American Association of Immunologists, Inc., This work was supported by Hungarian grants from the National Research Fund (K77587, TS-44798, and F67632).

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A(2A) receptor (A(2A)R), but its role in EC ROS production remains unknown. In this study, we investigated the role of A(2A)R in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4-10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A(2A)R. These were accompanied by p47(phox), ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A(2A)R antagonist (SCH58261), or adenosine deaminase, or by knockdown of A(2A)R or Nox2 using specific siRNAs. Knockdown of A(2A)R, as determined by Western blotting, decreased Nox2 and p47(phox) expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A(2A)R knock-out mice. In conclusion, A(2A)R is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A(2A)R protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Huntington's disease (HD) is a progressive neurodegenerative genetic disorder which leads to motor, cognitive and psychiatric disturbances. The primary neuropathological hallmark is atrophy of the striatum. Cannabinoid CB1 receptors (CB1Rs) are particularly enriched in the striatum and previous works indicate their early loss of expression in HD, even before symptom occurrence. However, pathophysiological significance of this loss of expression remains unclear. In addition, whether specific modulation of CB1R is able to mitigate striatal neuron fate in HD remains currently controversial. In order to gain further insights on the potential role of CB1R in HD physiopathology, we evaluated the pathophysiological consequences of a genetic deletion of CB1R in the N171-82Q transgenic model and following 3-nitropropionic (3NP) intoxication. Taken together our data demonstrate that CB1R knockout (1) worsens motor performances in N171-82Q mice and (2) increases mouse susceptibility to 3NP. These results suggest that functional changes in CB1R may contribute to the physiopathological development of HD.

Recent reports have shown that adenosine A 1 receptor-mediated cardioprotection requires concomitant A 2 receptor activation, but no study thus far has shown that this phenomenon occurs using A, agonists at reperfusion. Thus, we compared adenosine A 2A receptor knockout (A 2A KO) and wild-type mouse hearts (n = 9-11) subjected to global ischemia (30 minutes) and reperfusion (60 minutes) in the presence and absence of the A 1 agonist N 6 -cyclopentlyadenosine (CPA). We also determined the effects of selective antagonists at A 2A and A 2B receptors on CPA-induced protection. In wild-type hearts, CPA (100 nM) significantly (P < 0.05) improved contractility (52.7 ± 6.2% versus 23.9 ± 4.9% of preischemia), left ventricular developed pressure, end diastolic pressure; reduced infarct size (7.9 ± 1.7% versus 23.9 ± 6.6% area at risk); decreased lactate dehydrogenase efflux; and increased ERK1/2 phosphorylation at 60 minutes of reperfusion. Adenosine A 2A (ZM241385, 50 nM) and A 2B (MRS1754, 100 nM) receptor antagonists abolished CPA-mediated cardioprotection in wild-type groups as did the A 1 receptor antagonist DPCPX (P < 0.05). In A 2A KO hearts, CPA did not improve functional parameters and protective signaling with the exception of end diastolic pressure. In this model, using a clinically relevant mode of pharmacologic intervention, pERK 1/2-dependent A,-mediated cardioprotection requires a cooperative activation of A 2 receptors, presumably through endogenous adenosine.

Mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and A(2A) receptor antagonists have antinociceptive effects. We have previously shown a marked reduction in the behavioural responses to formalin injection in A(2A) receptor knockout mice. This may be due to the presence of pronociceptive A(2A) receptors on sensory nerves, and if so spinal cords from A(2A) receptor knockout mice may have altered neurochemical responses to a nociceptive stimulus. We tested this hypothesis by studying two parameters known to change with spinal cord activity, NMDA glutamate receptor binding and [(14)C]-2-deoxyglucose uptake, following intraplantar formalin injection in wild-type and A(2A) receptor knockout mice. In naïve untreated A(2A) knockout mice [(14)C]-2-deoxyglucose uptake in all regions of the spinal cord was significantly lower compared to the wild-type, similar to the reduced NMDA receptor binding that we have previously observed. Following formalin treatment, there was an decrease in [(3)H]-MK801 binding to NMDA receptors and an increase in [(14)C]-2-deoxyglucose uptake in the spinal cords of wild-type mice, and these changes were significantly reduced in the A(2A) knockout mice. In addition to altered behavioural responses, there are therefore corresponding reductions in spinal cord neurochemical changes induced by formalin in mice lacking adenosine A(2A) receptors. These observations support the hypothesis that activation of A(2A) receptors enhances nociceptive input into the spinal cord and suggests a possible role for A(2A) antagonists as analgesics.

During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile ("start-up"). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.

Adenosine mediates vascular responses through four receptor subtypes: A1, A2A, A2B, and A3. The role of A2A receptors in aortic vascular tone was investigated using A2A adenosine receptor (AR) knockout (A2AKO) and corresponding wild-type (A2AWT) mice. Isolated aortic rings from A2AWT and A2AKO mice were precontracted with phenylephrine (10−7 M), and concentration responses for adenosine analogs and selective agonists/antagonists were obtained. Nonselective adenosine analog (NECA; EC50 = 6.78 μM) and CGS-21680 (A2AAR selective agonist; EC50 = 0.013 μM) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at 10−5 M NECA and CGS-21680, respectively) in A2AWT aorta. In A2AKO aorta, NECA (EC50 = 0.075 μM) induced concentration-dependent contraction (maximum contraction of 47% at 10−6 M; P < 0.05 compared with A2AWT), whereas CGS-21680 produced no response. SCH-58261 (10−6 M; A2AAR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in A2AWT ( P < 0.05), whereas no change was observed in A2AKO. When DPCPX (10−5 M; A1 selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in A2AWT (50% vs. 25% in controls at 10−5 M; P < 0.05), whereas lower contraction was seen in A2AKO tissues (5% vs. 47% in controls at 10−6 M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60–6583 (A2B selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, A2AAR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A2AAR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism.

A long line of experimental evidence indicates that endogenous cannabinoid mechanisms play important roles in nociceptive information processing in various areas of the nervous system including the spinal cord. Although it is extensively documented that the cannabinoid-1 receptor (CB(1)-R) is strongly expressed in the superficial spinal dorsal horn, its cellular distribution is poorly defined, hampering our interpretation of the effect of cannabinoids on pain processing spinal neural circuits. Thus, we investigated the cellular distribution of CB(1)-Rs in laminae I and II of the rodent spinal dorsal horn with immunocytochemical methods. Axonal varicosities revealed a strong immunoreactivity for CB(1)-R, but no CB(1)-R expression was observed on dendrites and perikarya of neurons. Investigating the co-localization of CB(1)-R with markers of peptidergic and non-peptidergic primary afferents, and axon terminals of putative glutamatergic and GABAergic spinal neurons we found that nearly half of the peptidergic (immunoreactive for calcitonin gene-related peptide) and more than 20% of the non-peptidergic (binding isolectin B4) nociceptive primary afferents, more than one-third and approximately 20% of the axon terminals of putative glutamatergic (immunoreactive for vesicular glutamate transporter 2) and GABAergic (immunoreactive for glutamic acid decarboxylase; GAD65 and/or GAD67) spinal interneurons, respectively, were positively stained for CB(1)-R. In addition to axon terminals, almost half of the astrocytic (immunoreactive for glial fibrillary acidic protein) and nearly 80% of microglial (immunoreactive for CD11b) profiles were also immunolabeled for CB(1)-R. The findings suggest that the activity-dependent release of endogenous cannabinoids activates a complex signaling mechanism in pain processing spinal neural circuits into which both neurons and glial cells may contribute.

A(2A) adenosine receptor (A(2A)AR) has been shown to suppress superoxide generation in leukocytes via the cAMP-protein kinase A (PKA) pathway. However, no study has yet explored the role of A(2A)AR in relation to NADPH oxidase in murine tracheas in vitro, which may lead to altered smooth muscle relaxation in asthma. Therefore, the present study evaluated the effects of A(2A)AR deficiency on the NADPH oxidase pathway in tracheas of A(2A) wild-type (WT) and A(2A) knockout (KO) mice. A(2A)WT mice were sensitized with ovalbumin (30 microg i.p.) on days 1 and 6, followed by 5% ovalbumin aerosol challenge on days 11, 12, and 13. A(2A)AR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A(2A)WT sensitized mice compared with controls. A(2A)KO mice also showed decreased cAMP and p-PKA levels. A(2A)WT sensitized and A(2A)KO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A(2A)AR agonist, 4-[2-[[6-amino-9-(N-ethyl-beta-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A(2A)WT sensitized mice compared with the controls, although it was absent in A(2A)KO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A(2A)WT sensitized tracheas, whereas specific PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) blocked CGS 21680-induced relaxation. Tracheal reactive oxygen species (ROS) generation was also increased in A(2A)WT sensitized and A(2A)KO control mice compared with the controls. In conclusion, this study shows that A(2A)AR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.

Bursting activity of striatal medium spiny neurons results from membrane potential oscillations between a down- and an upstate that could be regulated by G-protein-coupled receptors. Among these, dopamine D(2) and adenosine A(2A) receptors are highly enriched in striatal neurons and exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. More particularly, respective involvements of common intracellular signaling cascades and A(2A)-D(2) receptor heteromerization remain unknown. Here we show, by performing perforated-patch-clamp recordings on brain slices and loading competitive peptides, that D(2) and A(2A) receptors regulate the induction by N-methyl-D-aspartate of a depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D(2) receptor activation abolished transitions between a hyperpolarized resting potential and a depolarized plateau potential by regulating the Ca(V)1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A(2A) receptor activation had no effect per se but fully reversed the effects of D(2) receptor activation through a mechanism in which A(2A)-D(2) receptors heteromerization is strictly mandatory, demonstrating therefore a first direct physiological relevance of these heteromers. Our results show that membrane potential transitions and firing patterns in striatal neurons are tightly controlled by D(2) and A(2A) receptors through specific protein-protein interactions including A(2A)-D(2) receptors heteromerization., In Vitro, Journal Article, Research Support, N.I.H. Intramural, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

International audience; The neuromodulator adenosine, acting through activation of four defined metabotropic receptors called A(1), 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(2A)R 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(2A)R KO) animals. A(2A)R 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.

The coding region of the herpes simplex type 1 virus thymidine kinase gene was coupled to the promoter of the bovine thyroglobulin gene and introduced into the genome of mice. The viral thymidine kinase (HSV1-TK) was expressed mainly in the thyroid glands and testis. Upon treatment of transgenic females with the antiherpetic agent Ganciclovir the thyroid regressed, while the parathyroid gland was unaffected. The number of thyroid follicle cells was greatly reduced after 3 days, and they were completely absent after 7 days of treatment. After 14 days, the levels of circulating T4 and T3 were below the limits of detection, total soluble protein recovered from the thyroid and parathyroid glands together was 10% of the control value, and the level of thyroid HSV1-TK was more than 100-fold lower than that in transgenic controls. Levels of circulating PTH and calcitonin remained normal. At the time of treatment the mice were adults. Thus, the thyroid follicle cells were selectively ablated after normal development with a functional thyroid gland. When treatment with Ganciclovir was terminated after 14 days, no circulating T4 or T3 or other indications of thyroid regeneration were detected for a subsequent period of 90 days. During this time the mice gained weight more slowly than controls, at a rate consistent with the suppression of GH synthesis by thyroid deficiency. The production of mouse major urinary protein (MUP) ceased in the treated mice and was completely restored by the administration of T4. MUP production was not restored by GH, demonstrating that the expression of the Mup genes requires T4 in addition to GH.

A hybrid gene comprising the bovine thyroglobulin gene promoter and the coding region for the simian virus-40 large T- and small t-antigens was used to generate 30 transgenic mice by microinjection into the pronuclei of single cell embryos. All animals except three developed, as single primitive pathology, a dramatic enlargement of the thyroid gland. Compression of trachea and esophagus, accompanied by dyspnea, inspiratory stridor, and dysphagia, led to a progressive cachexia and premature death attributed to respiratory failure. Despite the large thyroid volume, T4 levels were abnormally low, and the progression of the syndrome could be delayed by a substitutive treatment with thyroid hormones. The rapid evolution of the disease, leading to the death of most founder transgenic animals before the breeding age, prevented transmission of the transgene to their offspring. Only two transgenic lines are presently surviving. Immunohistochemical analysis of the tissues revealed a specific expression of the simian virus-40 antigens in the thyroid cells. Hyperplasia was already obvious at birth. Older animals displayed moderately to poorly differentiated thyroid adenocarcinomas. Electron microscopy revealed, however, the persistence of cell polarity and the presence of microfollicles between the densely packed cells. Cell lines derived from these large T-expressing thyroids were shown to have lost expression of both thyroglobulin and thyroperoxidase, while expressing low levels of TSH receptors. These transgenic mice could constitute an interesting model of aggressive adenocarcinoma, sharing phenotypical similarities with the anaplastic type of human thyroid tumors.

Motor impairment, dopamine (DA) neuronal activity and proenkephalin (PENK) gene expression in the caudate-putamen (CPu) were measured in 6-OHDA-lesioned and treated (l-DOPA. +. benserazide) CB1 KO and WT mice. A lesion induced by 6-OHDA produced more severe motor deterioration in CB1 KO mice accompanied by more loss of DA neurons and increased PENK gene expression in the CPu. Oxidative/nitrosative and neuroinflammatory parameters were estimated in the CPu and cingulate cortex (Cg). CB1 KO mice exhibited higher MDA levels and iNOS protein expression in the CPu and Cg compared to WT mice. Treatment with l-DOPA. +. benserazide (12 weeks) resulted in less severe dyskinesias in CB1 KO than in WT mice.The results revealed that the lack of cannabinoid CB1 receptors increased the severity of motor impairment and DA lesion, and reduced l-DOPA-induced dyskinesias. These results suggest that activation of CB1 receptors offers neuroprotection against dopaminergic lesion and the development of l-DOPA-induced dyskinesias. © 2009 Elsevier Inc., This research was supported by grants from “FUNDACIÓN la CAIXA” and “Fundación de Neurociencias y Envejecimiento” to JM, the “CATEDRADISTEC” for research in Parkinson’s disease (JM and CL) and to CAM S-SAL/0261/2006 (JCL).SPR was a predoctoral fellow supported by “Fundación la CAIXA”. MSGG is a predoctoral fellow of the Ministry of Health. BGPN is a MEC-FPU program fellow.

International audience; We have shown previously that mice lacking the adenosine A2A receptor (A2AR) generated on a CD1 background self-administer more ethanol and exhibit hyposensitivity to acute ethanol. We aimed to investigate if the increased propensity of A2A(-/-) mice to consume ethanol is associated with an altered sensitivity in the motivational properties of ethanol in the conditioned place preference (CPP) and conditioned taste aversion (CTA) paradigms and with an altered development of sensitization to the locomotor effects of ethanol. We also tested their sensitivity to the anxiolytic effects of ethanol. Our results show that A2A(-/-) mice produced on a CD1 background displayed a reduced ethanol-induced CPP and an increased sensitivity to the anxiolytic and locomotorstimulant effects of ethanol, but they did not show alteration in ethanol-induced CTA and locomotor sensitization. Ethanol-induced CPP, ethanol consumption and the locomotor effects of ethanol were also tested in A2A(-/-) mice produced on a C57BL/6J background. Our results emphasized the importance of the genetic background because alteration in ethanol consumption and preference, ethanol-induced CPP and locomotor-stimulant effects were not found in knockout mice produced on the alcohol-preferring C57BL/6J genetic background. Finally, the A2AR agonist, 2-p-(2-carboxyethyl)-phenylethylamino-50-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680), reduced ethanol consumption and preference in C57BL/6J mice. In conclusion, A2AR deficiency in mice generated on a CD1 background leads to high ethanol consumption that is associated with an increased sensitivity to the locomotor-stimulant/anxiolytic effects of ethanol and a decrease in ethanol-induced CPP.

Regulation of Ca(2+) homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB(1) and CB(2) receptors have been shown to regulate Ca(2+) levels and/or K(+) currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca(2+) influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K(+) concentration (50 mM). The CB(1) receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration-dependent inhibition of depolarization-evoked Ca(2+) transients in oligodendroglial somata with a maximal effect (94+/-3)% and an EC(50) of 1.3+/-0.03 microM. This activity was mimicked by the CB(1)/CB(2) agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB(2) receptor selective agonist JWH133 was ineffective. The CB(1) receptor antagonist AM251 (1 microM) also reduced the Ca(2+) response evoked by high extracellular K(+) and did not prevent the inhibition elicited by ACEA (3 microM). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca(2+) transients was significantly reduced in oligodendrocytes from CB(1) receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K(+) channels (Kir channels) blockers BaCl(2) (300 microM) and CsCl(2) (1 mM) reduced the size of voltage-induced Ca(2+) influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca(2+) transients in oligodendrocytes via CB(1) receptor-independent and -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels.

We hypothesized that A2Aadenosine receptor (A2AAR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2AAR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5′- N-ethylcarboxamide (NECA; 10−6M), an adenosine analog, caused relaxation in wild-type A2AAR (A2AAR+/+; +33.99 ± 4.70%, P < 0.05) versus contraction in A2AAR knockout (A2AAR−/−; −27.52 ± 4.11%) mouse aortae. An A2AAR-specific antagonist (SCH-58261; 1μM) changed the NECA (10−6M) relaxation response to contraction (−35.82 ± 4.69%, P < 0.05) in A2AAR+/+aortae, whereas no effect was noted in A2AAR−/−aortae. Significant contraction was seen in the absence of the endothelium in A2AAR+/+(−2.58 ± 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor ( N-nitro-l-arginine methyl ester; 100 μM) and a cyclooxygenase inhibitor (indomethacin; 10 μM) failed to block NECA-induced relaxation in A2AAR+/+aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 μM) changed NECA-mediated relaxation (−22.74 ± 5.11% at 10−6M) and CGS-21680-mediated relaxation (−18.54 ± 6.06% at 10−6M) to contraction in A2AAR+/+aortae, whereas no response was noted in A2AAR−/−aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5( Z)-enoic acid; 10 μM] was able to block NECA-induced relaxation in A2AAR+/+aortae, whereas ω-hydroxylase inhibitors (10 μM dibromo-dodecenyl-methylsulfimide and 10 μM HET-0016) changed contraction into relaxation in A2AAR−/−aorta. Cyp2c29 protein was upregulated in A2AAR+/+aortae, whereas Cyp4a was upregulated in A2AAR−/−aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2AAR+/+versus A2AAR−/−aortae. EET levels were not significantly different between A2AAR+/+and A2AAR−/−aortae. It is concluded that CYP epoxygenases play an important role in A2AAR-mediated relaxation, and the deletion of the A2AAR leads to contraction through Cyp4a.

GPR3 is an orphan G protein-coupled receptor endowed with constitutive Gs signaling activity, which is expressed broadly in the central nervous system, with maximal expression in the habenula. We investigated the consequences of its genetic deletion in several behavioral paradigms and on neurotransmission. Compared to wild-type, hippocampal neurons from Gpr3(-/-) mice displayed lower basal intracellular cAMP levels, consistent with the strong constitutive activity of GPR3 in transiently transfected cells. Behavioral analyses revealed that Gpr3(-/-) mice exhibited a high level of avoidance of novel and unfamiliar environment, associated with increased stress reactivity in behavioral despair paradigms and aggressive behavior in the resident-intruder test. On the contrary, no deficit was found in the learning ability to avoid an aversive event in active avoidance task. The reduced ability of Gpr3(-/-) mice to cope with stress was unrelated to dysfunction of the hypothalamic-pituitary-adrenal axis, with Gpr3(-/-) mice showing normal corticosterone production under basal or stressful conditions. In contrast, dramatic alterations of monoamine contents were found in hippocampus, hypothalamus and frontal cortex of Gpr3(-/-) mice. Our results establish a link between tonic stimulation of the cAMP signaling pathway by GPR3 and control of neurotransmission by monoamines throughout the forebrain. GPR3 qualifies as a new player in the modulation of behavioral responses to stress and constitutes a novel promising pharmacological target for treatment of emotional disorders., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Endocannabinoids are lipidic modulators able to bind cannabinoid receptors (CNRs). Two types of CNRs have been cloned, CNR1 (central) and CNR2 (peripheral). The objectives of the present study were to investigate the expression pattern of CNR1 in the rat testis during prepubertal development and to define the CNR1 spatiotemporal pattern. From 31 to 60 days of age, CNR1 was immunolocalized in round elongating spermatids and spermatozoa, suggesting an important role for this receptor in spermatogenesis. From 14 to 60 days of age, adult Leydig cells (ALCs) at different developmental stages were positive for CNR1. In particular, CNR1 expression in differentiating ALCs was negatively correlated to cell division. Bromodeoxyuridine uptake experiments on serial sections showed that immature Leydig cells in mitosis were negative for CNR1; in contrast, immature nonmitotic Leydig cells were positive for CNR1. A further observation of few ALCs in CNR1KO mice validates the role of CNR1 during proliferative activity involved in ALC differentiation. In addition, starting from 41 days of age, a faint CNR1 signal was also observed in Sertoli cells. Taken together, these results demonstrate the first clear evidence (to our knowledge) of CNR1 in mammalian germinal epithelium, ALCs, and Sertoli cells and indicate that differentiation of ALCs may depend on the endocannabinoid system.

Adenosine triphosphate has previously been shown to induce semi-mature human monocyte-derived dendritic cells (DC). These are characterized by the up-regulation of co-stimulatory molecules, the inhibition of IL-12 and the up-regulation of some genes involved in immune tolerance, such as thrombospondin-1 and indoleamine 2,3-dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y(11) receptor; since there is no functional P2Y(11) gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5'-(3-thiotriphosphate) and adenosine inhibited the production of IL-12p70 by bone marrow-derived DC (BMDC). These inhibitions were relieved by 8-p-sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A(2B) (-/-) BMDC indicated the involvement of the A(2B) receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5'-(N-ethylcarboxamido) adenosine (NECA, the most potent A(2B) receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin-1 and vascular endothelial growth factor were up-regulated whereas CCL2 and CCL12 were down-regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A(2B) receptor, and will possibly lead to an impairment of Th1 response or tolerance.

Peripheral nerve injury produces a persistent neuropathic pain state characterized by spontaneous pain, allodynia and hyperalgesia. In this study, we evaluated the possible involvement of A 2ARs in the development of neuropathic pain and the expression of microglia and astrocytes in the spinal cord after sciatic nerve injury. For this purpose, partial ligation of the sciatic nerve was performed in A 2A knockout mice and wild-type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold-plate test and the plantar test, respectively. In wild-type animals, sciatic nerve injury led to a neuropathic pain syndrome that was revealed in these three nociceptive behavioural tests. However, a significant decrease of the mechanical allodynia and a suppression of thermal hyperalgesia and allodynia were observed in A 2AR deficient mice. The expression of microglia and astrocytes was enhanced in wild-type mice exposed to sciatic nerve injury and this response was attenuated in knockout animals. Taken together, our results demonstrate the involvement of A 2ARs in the control of neuropathic pain and propose this receptor as an interesting target for the development of new drugs for the management of this clinical syndrome.

We investigated the effect of a synthetic cannabinoid, WIN 55,212-2 on excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells. Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 microM. In rats and mice, this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and muM concentrations. When the function of CB(1) cannabinoid receptors (CB(1)R) was impaired, either by the application of a CB(1)R antagonist AM251, or by using CB(1)R knockout mice, WIN 55,212-2 in microM concentrations could still significantly reduced the amplitude of EPSCs. WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites, since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased. The inactive enantiomer, WIN 55,212-3, mimicked the effect of WIN 55,212-2 applied in high doses. In further experiments we found that the CB(1)R-independent effect of 10 microM WIN 55,212-2 at glutamatergic synapses was fully abolished, when slices were pre-treated with omega-conotoxin GVIA, but not with omega-agatoxin IVA. These data suggest that, in the hippocampus, WIN 55,212-2 reduces glutamate release from Schaffer collaterals solely via CB(1)Rs in the nM concentration range, whereas in microM concentrations, WIN 55,212-2 suppresses excitatory transmission, in addition to activation of CB(1)Rs, by directly blocking N-type voltage-gated Ca(2+) channels independent of CB(1)Rs., In Vitro, Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A(2A) genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A(2A) AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A(2A) AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A(2B) AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A(2A) AR expression was absent in A2AKO. There was no difference in A(1) and A(3) ARs between WT and KO animals. The concentration-relaxation curve for adenosine-5'-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration-response curve for A(2B) selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 microM in A2AWT (76.32+/-11.35% from baseline, n=5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 microM, 93.32+/-5.8% from baseline, n=5). L-NMA (1 microM, n=4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66+/-3.23% from baseline in A2AWT, while 81.76+/-8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 microM NECA was significantly blunted with 10 microM L-NAME (377.08+/-25.23% to 305.41+/-30.73%, n=9) in A2AWT but not in A2AKO (153.66+/-22.7% to 143.88+/-36.65%, n=5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346+/-22.85 to 277+/-31.39, n=6). No change in CF to CGS-21680 was noted in A(2A)AKO. Our data demonstrate, for the first time, that coronary A(2B) AR was up-regulated in mice deficient in A(2A) AR. We also provide direct evidence supporting a role for NO in A(2A) AR-mediated coronary vasodilation. The data further support the role for A(2A) AR in the regulation of basal coronary tone through the release of NO.

Cannabinoids may exhibit symptom control in multiple sclerosis (MS). We show here that cannabinoid receptor (CBR) agonists can also be immunosuppressive and neuroprotective in models of MS. Immunosuppression was associated with reduced: myelin-specific T cell responses; central nervous system infiltration and reduced clinical disease. This was found to be largely CB(1)R-dependent and only occurred at doses that induced significant cannabimimetic effects that would not be achieved clinically. Lower, non-immunosuppressive doses of cannabinoids however, slowed the accumulation of nerve loss and disability, despite failing to inhibit relapses. This further highlights the neuroprotective potential of cannabinoids to slow the progression of MS.

International audience; 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.

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (POC), yet it is unclear which adenosine receptor subtype is primarily responsible. To evaluate the role of A2A adenosine receptors in POC-induced protection, global ischemia-reperfusion was performed with and without POC in isolated wild-type (WT) and A2A adenosine receptor knockout (A2AKO) mouse hearts. Injury was measured in terms of postischemic functional recovery and release of cardiac troponin I (cTnI). Activation of protective signaling with POC was assessed by Akt and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In WT hearts, POC improved recovery of postischemic developed pressure in early (81.6 ± 6.4% of preischemic baseline vs. 37.5 ± 5.6% for non-POC WT at 1 min) and late (62.2 ± 4.2% of baseline vs. 45.5 ± 5.3% for non-POC WT at 30 min) reperfusion, reduced cTnI release by 37%, and doubled the phosphorylation of both Akt and ERK1/2. These beneficial effects of POC were blocked by treatment with the selective A2A adenosine receptor antagonist ZM-241385 during reperfusion. Postischemic functional recovery, cTnI release, and phosphorylation of Akt and ERK1/2 were not different between non-POC WT and A2AKO hearts. In A2AKO hearts, POC did not improve functional recovery, reduce cTnI release, nor increase phosphorylation of Akt or ERK1/2. Thus the protective effects of POC are attenuated by both selective A2A receptor antagonism and targeted deletion of the gene encoding A2A adenosine receptors. These observations support the conclusion that endogenous activation of A2A adenosine receptors is an essential trigger leading to the protective effects of POC in isolated murine hearts.

In the present study we investigated whether serotonin release in the hippocampus is subject to regulation via cannabinoid receptors. Both rat and mouse hippocampal slices were preincubated with [3H]serotonin ([3H]5-HT) and superfused with medium containing serotonin reuptake inhibitor citalopram hydrobromide (300 nM). The cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2, 1 microM) did not affect either the resting or the electrically evoked [3H]5-HT release. In the presence of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP-5, 50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione-disodium (CNQX, 10 microM) the evoked [3H]5-HT release was decreased significantly. Similar findings were obtained when CNQX (10 microM) was applied alone with WIN55,212-2. This effect was abolished by the selective cannabinoid receptor subtype 1 (CB1) antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716, 1 microM) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251, 1 microM). Similarly to that observed in rats, WIN55,212-2 (1 microM) decreased the evoked [3H]5-HT efflux in wild-type mice (CB1+/+). The inhibitory effect of WIN55,212-2 (1 microM) was completely absent in hippocampal slices derived from mice genetically deficient in CB1 cannabinoid receptors (CB1-/-). Relatively selective degeneration of fine serotonergic axons by the neurotoxin parachloramphetamine (PCA) reduced significantly the tritium uptake and the evoked [3H]5-HT release. In addition, PCA, eliminated the effect of WIN55,212-2 (1 microM) on the stimulation-evoked [3H]5-HT efflux. In contrast to the PCA-treated animals, WIN55,212-2 (1 microM) reduced the [3H]5-HT efflux in the saline-treated group. Our data suggest that a subpopulation of non-synaptic serotonergic afferents express CB1 receptors and activation of these CB1 receptors leads to a decrease in 5-HT release.

The endocannabinoid CB(1) receptor has been implicated in the inhibitory control of learning and memory. In the present experiment, we compared the behavioral response of CB(1) receptor knockout mice (CB(1)R(-/-)) with animals administered CB(1) receptor antagonist/inverse agonist SR141716A (rimonabant; 3 mg/kg IP, 30 min pre-trial) in terms of acquisition and retention of a habituation task and changes in cerebral monoamines. The results can be summarized as follows: (i) the acute and chronic invalidation of the CB(1) receptor resulted in an increase of behavioral habituation during the first exposure to an open field, indicative of enhanced acquisition of the task; (ii) CB(1)R(-/-) mice, but not rimonabant-treated animals, showed enhanced retention of the habituation task when re-tested 48 h and 1 week subsequent to the first exposure to the open field, respectively; (iii) the facilitation of retention of the habituation task in CB(1)R(-/-) mice was accompanied by a selective and site-specific increase in serotonin activity in hippocampus; and (iv) rimonabant-treated animals displayed 'antidepressant-like' neurochemical alterations of cerebral monoamines, that is, most parameters of monoaminergic activity were increased especially in dorsal striatum and hippocampus. Taken together, the present findings demonstrate that the genetic disruption of the CB(1) receptor gene can cause an improvement of behavioral habituation, which is considered to represent a form of 'non-associative' learning. Furthermore, our data support the assumption of a rimonabant-sensitive cannabinoid receptive site that is different from the 'classical' CB(1) receptor and which, under physiological conditions, might be involved in the inhibitory control of the acquisition but not retention of non-associative learning tasks.

Background 3,4-Methylenedioxymethamphetamine (MDMA) is a popular recreational drug widely abused by young people. The endocannabinoid system is involved in the addictive processes induced by different drugs of abuse. However, the role of this system in the pharmacological effects of MDMA has not yet been clarified. Methods Locomotion, body temperature, and anxiogenic-like responses were evaluated after acute MDMA administration in CB 1 cannabinoid receptor 1 knockout mice. Additionally, MDMA rewarding properties were investigated in the place conditioning and the intravenous self-administration paradigms. Extracellular levels of dopamine (DA) in the nucleus accumbens were also analyzed after a single administration of MDMA by in vivo microdialysis. Results Acute MDMA administration increased locomotor activity, body temperature, and anxiogenic-like responses in wild-type mice, but these responses were lower or abolished in knockout animals. 3,4-Methylenedioxymethamphetamine produced similar conditioned place preference and increased dopamine extracellular levels in the nucleus accumbens in both genotypes. Nevertheless, CB 1 knockout mice failed to self-administer MDMA at any of the doses used. Conclusions These results indicate that CB 1 cannabinoid receptors play an important role in the acute prototypical effects of MDMA and are essential in the acquisition of an operant behavior to self-administer this drug.