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Sample records for a2 txa2 receptor

  1. The enhancement of TXA2 receptors-mediated contractile response in intrarenal artery dysfunction in type 2 diabetic mice.

    PubMed

    Kuang, Su-Juan; Qian, Jie-Sheng; Yang, Hui; Rao, Fang; Chen, Xiao-Yan; Zhang, Meng-Zhen; Shan, Zhi-Xin; Lin, Qiu-Xiong; Xue, Yu-Mei; Wu, Shu-Lin; Jiang, Li; Chen, Chun-Bo; Deng, Chun-Yu

    2017-03-09

    Thromboxane A2 (TXA2) has been implicated in the pathogenesis of diabetic vascular complications, although the underlying mechanism remains unclear. The present study investigated the alterations in TXA2 receptor signal transduction in type 2 diabetic renal arteries. The contraction of renal arterial rings in control (db/m+) mice and type 2 diabetic (db/db) mice was measured by a Multi Myograph System. Intracellular calcium concentration ([Ca(2+)]i) in vascular smooth muscle cells was measured by Fluo-4/AM dye and confocal laser scanning microscopy. Quantitative real-time PCR and Western blot analysis were used to determine gene and protein expression levels, respectively. A stable TXA2 mimic U46619 caused markedly stronger dose-dependent contractions in the renal arteries of db/db mice than in those of db/m(+) mice. This response was completely blocked by a TXA2 receptor antagonist GR32191 and significantly inhibited by U73122. U46619-induced vasoconstriction was increased in the presence of nifedipine in db/db mice compared with that in db/m(+) mice, whereas the response to U46619 did not differ between the two groups in the presence of SKF96365. Sarcoplasmic reticulum Ca(2+) release-mediated and CaCl2-induced contractions did not differ between the two groups. In db/db mice, store-operated Ca(2+) (SOC) entry-mediated contraction in the renal arteries and SOC entry-mediated Ca(2+) influx in smooth muscle cells were significantly increased. And the gene and protein expressions of TXA2 receptors, Orai1 and Stim1 were upregulated in the diabetic renal arteries. Therefore the enhancement of U46619-induced contraction was mediated by the upregulation of TXA2 receptors and downstream signaling in the diabetic renal arteries.

  2. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

    PubMed Central

    Thomas, D W; Mannon, R B; Mannon, P J; Latour, A; Oliver, J A; Hoffman, M; Smithies, O; Koller, B H; Coffman, T M

    1998-01-01

    Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus. PMID:9835625

  3. Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

    PubMed Central

    Golino, P; Rosolowsky, M; Yao, S K; McNatt, J; De Clerck, F; Buja, L M; Willerson, J T

    1990-01-01

    We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1

  4. Thromboxane A2 receptor antagonist SQ29548 reduces ischemic stroke-induced microglia/macrophages activation and enrichment, and ameliorates brain injury

    PubMed Central

    Yan, Aijuan; Zhang, Tingting; Yang, Xiao; Shao, Jiaxiang; Fu, Ningzhen; Shen, Fanxia; Fu, Yi; Xia, Weiliang

    2016-01-01

    Thromboxane A2 receptor (TXA2R) activation is thought to be involved in thrombosis/hemostasis and inflammation responses. We have previously shown that TXA2R antagonist SQ29548 attenuates BV2 microglia activation by suppression of ERK pathway, but its effect is not tested in vivo. The present study aims to explore the role of TXA2R on microglia/macrophages activation after ischemia/reperfusion brain injury in mice. Adult male ICR mice underwent 90-min transient middle cerebral artery occlusion (tMCAO). Immediately and 24 h after reperfusion, SQ29548 was administered twice to the ipsilateral ventricle (10 μl, 2.6 μmol/ml, per dose). Cerebral infarction volume, inflammatory cytokines release and microglia/macrophages activation were measured using the cresyl violet method, quantitative polymerase chain reaction (qPCR), and immunofluorescence double staining, respectively. Expression of TXA2R was significantly increased in the ipsilateral brain tissue after ischemia/reperfusion, which was also found to co-localize with activated microglia/macrophages in the infarct area. Administration of SQ29548 inhibited microglia/macrophages activation and enrichment, including both M1 and M2 phenotypes, and attenuated ischemia-induced IL-1ß, IL-6, and TNF-α up-regulation and iNOS release. TXA2R antagonist SQ29548 inhibited ischemia-induced inflammatory response and furthermore reduced microglia/macrophages activation and ischemic/reperfusion brain injury. PMID:27775054

  5. Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

    SciTech Connect

    Dorn, G.W. II; De Jesus, A. )

    1989-12-01

    To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of (125I)BOP was studied. (125I)BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced (125I)BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that (125I)BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.

  6. Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo

    SciTech Connect

    Shin, Y.; Romstedt, K.J.; Doyle, K.; Harrold, M.W.; Gerhardt, M.A.; Miller, D.D.; Patil, P.N.; Feller, D.R. )

    1991-01-01

    Although (-)-(S)-trimetoquinol (1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ) is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of (3H)SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.

  7. Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPβ isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

    PubMed

    O'Sullivan, Aine G; Mulvaney, Eamon P; Kinsella, B Therese

    2017-04-01

    The prostanoid thromboxane (TX) A2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA2/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA2-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA2/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA2/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA2/TP signalling axis in PCa, including potentially in CRPC.

  8. Thromboxane A2, released by the anti-tumour drug irinotecan, is a novel stimulator of Cl- secretion in isolated rat colon.

    PubMed Central

    Sakai, H; Sato, T; Hamada, N; Yasue, M; Ikari, A; Kakinoki, B; Takeguchi, N

    1997-01-01

    1. A camptothecin derivative, irinotecan (Cpt-11), is a topoisomerase I inhibitor and has a strong activity against a broad range of human cancer. One of the side-effects of this drug is diarrhoea. Here, we tried to determine the mediator of the irinotecan-induced Cl- secretion which may underlie this diarrhoea, using isolated mucosae of rat distal colon. 2. Irinotecan increased Cl- secretory current in a concentration-dependent manner across the mucosa, set between Ussing chambers. Thromboxane A2 (TXA2) has not been reported to date as a physiological stimulant of Cl- secretion in the distal colon. However, the major part of the present irinotecan-induced current was inhibited by selective thromboxane A2 receptor antagonists (KW-3635 and ONO-3708), and a selective thromboxane synthase inhibitor (Y-20811). In fact, we found that irinotecan stimulated the release of TXA2 in a concentration-dependent manner from the isolated mucosa into the bathing solutions. 3. Furthermore, 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, induced Cl- secretion, which was almost completely inhibited by the TXA2 receptor antagonists. 4. In single cells of isolated crypts, STA2 depolarized the cell and increased the membrane conductance, indicating that STA2 opened the apical Cl- channel of the crypt cells. 5. We conclude, therefore, that the irinotecan-induced endogenous TXA2 is a novel stimulant of the Cl- secretion from the crypt cells of distal colon. PMID:9409477

  9. Thromboxane A2 exacerbates acute lung injury via promoting edema formation

    PubMed Central

    Kobayashi, Koji; Horikami, Daiki; Omori, Keisuke; Nakamura, Tatsuro; Yamazaki, Arisa; Maeda, Shingo; Murata, Takahisa

    2016-01-01

    Thromboxane A2 (TXA2) is produced in the lungs of patients suffering from acute lung injury (ALI). We assessed its contribution in disease progression using three different ALI mouse models. The administration of hydrochloric acid (HCl) or oleic acid (OA)+ lipopolysaccharide (LPS) caused tissue edema and neutrophil infiltration with TXA2 production in the lungs of the experimental mice. The administration of LPS induced only neutrophil accumulation without TXA2 production. Pretreatment with T prostanoid receptor (TP) antagonist attenuated the tissue edema but not neutrophil infiltration in these models. Intravital imaging and immunostaining demonstrated that administration of TP agonist caused vascular hyper-permeability by disrupting the endothelial barrier formation in the mouse ear. In vitro experiments showed that TP-stimulation disrupted the endothelial adherens junction, and it was inhibited by Ca2+ channel blockade or Rho kinase inhibition. Thus endogenous TXA2 exacerbates ALI, and its blockade attenuates it by modulating the extent of lung edema. This can be explained by the endothelial hyper-permeability caused by the activation of TXA2-TP axis, via Ca2+- and Rho kinase-dependent signaling. PMID:27562142

  10. Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane-mediated thromboxane A2 receptor activation.

    PubMed

    Bauer, Jochen; Ripperger, Anne; Frantz, Stefan; Ergün, Süleyman; Schwedhelm, Edzard; Benndorf, Ralf A

    2014-07-01

    Isoprostanes are free radical-catalysed PG-like products of unsaturated fatty acids, such as arachidonic acid, which are widely recognized as reliable markers of systemic lipid peroxidation and oxidative stress in vivo. Moreover, activation of enzymes, such as COX-2, may contribute to isoprostane formation. Indeed, formation of isoprostanes is considerably increased in various diseases which have been linked to oxidative stress, such as cardiovascular disease (CVD), and may predict the atherosclerotic burden and the risk of cardiovascular complications in the latter patients. In addition, several isoprostanes may directly contribute to the functional consequences of oxidant stress via activation of the TxA2 prostanoid receptor (TP), for example, by affecting endothelial cell function and regeneration, vascular tone, haemostasis and ischaemia/reperfusion injury. In this context, experimental and clinical data suggest that selected isoprostanes may represent important alternative activators of the TP receptor when endogenous TxA2 levels are low, for example, in aspirin-treated individuals with CVD. In this review, we will summarize the current understanding of isoprostane formation, biochemistry and (patho) physiology in the cardiovascular context.

  11. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  12. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  13. Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor

    PubMed Central

    Cong, Yue; Wang, Limei; Peng, Renjun; Zhao, Yang; Bai, Fan; Yang, Chao; Liu, Xiaolan; Wang, Daqian; Ma, Baiping; Cong, Yuwen

    2016-01-01

    The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders. PMID:27934923

  14. Thromboxane A2 Regulates CXCL1 and CXCL8 Chemokine Expression in the Nasal Mucosa–Derived Fibroblasts of Chronic Rhinosinusitis Patients

    PubMed Central

    Tsai, Yih-Jeng; Hao, Sheng-Po; Chen, Chih-Li; Wu, Wen-Bin

    2016-01-01

    Background Chronic rhinosinusitis without nasal polyps (CRSsNP) is a common chronic disease and the etiology remains unclear. Thromboxane A2 (TXA2) participates in platelet aggregation and tissue inflammation. In this study, the CXCL1/8 chemokine and TXA2-TP receptor expression in the CRSsNP mucosa was investigated. Experimental Approach Immunohistochemistry, chemokine release assay by ELISA, RT-PCR, Real-time PCR, Western blotting, pharmacological and siRNA knockdown analysis were applied in the CRSsNP tissue specimen and cultured nasal mucosa-derived fibroblasts. Results The immunohistochemistry results indicated that CXCL1 and CXCL8 were highly expressed in the CRSsNP mucosa compared with the controls; however, the TP receptors were expressed in both mucosa. Therefore, U46619 and IBOP, a TXA2 analog and TP agonist, were used to explore the role of TP activation in CXCL1/8 expression; both of these induced CXCL1/8 mRNA and protein expression in CRSsNP mucosa-derived fibroblasts. U46619 phosphorylated PI-3K, cyclic AMP (cAMP)/PKA, PKC, and cAMP response element (CREB). Activation of cAMP/PKA, PKC, and CREB was the major pathway for cxcl1/8 gene transcription. Pharmacological and siRNA knockdown analyses revealed that activation of cAMP/PKA and PKCμ/PKD pathways were required for CREB phosphorylation and PKA/C crosstalked with the PI-3K pathway. Conclusion and Implications Our study provides the first evidence for abundant TP receptor and CXCL1/8 expression in human CRSsNP mucosa and for TXA2 stimulation inducing CXCL1/8 expression in nasal fibroblasts primarily through TP receptor, cAMP/PKA, PKCμ/PKD, and CREB-related pathways. PMID:27351369

  15. Restoration of blood pressure by centrally injected U-46619, a thromboxane A(2) analog, in hemorrhaged hypotensive rats: investigation of different brain areas.

    PubMed

    Yalcin, Murat; Savci, Vahide

    2004-04-01

    In the present study, we investigated the cardiovascular effects of centrally injected U-46619, a thromboxane A(2) (TXA(2)) analog, and the central and peripheral mechanisms of these effects in hemorrhagic shock conditions. Hemorrhage was performed by withdrawing a total volume of 2.1 ml of blood/100 g body weight over a period of 10 min. Injections were made into the lateral cerebral ventricle (LCV), nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVLM) and paraventricular nucleus of hypothalamus (PVN). U-46619 (0.1, 1 and 2 microg) increased blood pressure and reversed hypotension in hemorrhagic shock. The pressor effect was dose- and time-dependent in all investigated brain areas. Heart rate changes were not significantly different in all groups. Pretreatment of rats with an injection of SQ-29548 (4 or 8 microg), a TXA(2) receptor antagonist, into the LCV, NTS, RVLM and PVN completely blocked the pressor effect of U-46619 (1 microg) injected into respective brain areas. Hemorrhage itself increased plasma adrenaline, noradrenaline, vasopressIN levels and renin activity. U-46619 (1 microg) injected into the LCV, PVN, RVLM and NTS produced additional increases in these hormone levels and in renin activity. Intravenous pretreatments of rats with prazosin (0.5 mg/kg), an alpha(1)-adrenoceptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylenepropionyl(1), O-Me-Tyr(2),Arg(8)]- vasopressin (10 microg/kg), a vasopressin V(1)-receptor antagonist, or saralasin (250 microg/kg), an angiotensin II receptor antagonist, in hemorrhaged rats partially blocked the pressor response to U-46619 (1 microg) injected into the LCV, PVN, RVLM and NTS. Results show that centrally administered U-46619, a TXA(2) analog, increases blood pressure and reverses hypotension in hemorrhagic shock. Activation of central TXA(2) receptors mediates the pressor effect of the drug. Furthermore, the increases in plasma adrenaline, noradrenaline, vasopressin levels and renin

  16. Hypertension increases contractile responses to hydrogen peroxide in resistance arteries through increased thromboxane A2, Ca2+, and superoxide anion levels.

    PubMed

    García-Redondo, Ana Belén; Briones, Ana María; Beltrán, Amada Elia; Alonso, María Jesús; Simonsen, Ulf; Salaices, Mercedes

    2009-01-01

    This study investigated the mechanisms underlying the response to hydrogen peroxide (H(2)O(2)) in mesenteric resistance arteries from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto (WKY) rats. Arteries were mounted in microvascular myographs for isometric tension recording and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), superoxide anion (O(2)(.)) production was evaluated by dihydroethidium fluorescence and confocal microscopy, and thromboxane A(2) (TXA(2)) production was evaluated by enzyme immunoassay. H(2)O(2) (1-100 microM) induced biphasic responses characterized by a transient endothelium-dependent contraction followed by relaxation. Simultaneous measurements of tension and Ca(2+) showed a greater effect of H(2)O(2) in arteries from hypertensive than normotensive rats. The cyclooxygenase (cox) inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 microM); the COX-1 inhibitor, SC-58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole] (1 microM); the thromboxane (TXA(2)) synthase inhibitor, furegrelate [5-(3-pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt] (10 microM); and the TXA(2)/prostaglandin H(2) receptor antagonist, SQ 29,548 ([1S-[1.alpha.,2.alpha.(Z),3.alpha.,4.alpha.

  17. Cordycepin-Enriched WIB801C from Cordyceps militaris Inhibits Collagen-Induced [Ca(2+)]i Mobilization via cAMP-Dependent Phosphorylation of Inositol 1, 4, 5-Trisphosphate Receptor in Human Platelets.

    PubMed

    Lee, Dong-Ha; Kim, Hyun-Hong; Cho, Hyun-Jeong; Yu, Young-Bin; Kang, Hyo-Chan; Kim, Jong-Lae; Lee, Jong-Jin; Park, Hwa-Jin

    2014-05-01

    In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca(2+)]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca(2+)]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca(2+)]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca(2+)-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

  18. Cordycepin-Enriched WIB801C from Cordyceps militaris Inhibits Collagen-Induced [Ca2+]i Mobilization via cAMP-Dependent Phosphorylation of Inositol 1, 4, 5-Trisphosphate Receptor in Human Platelets

    PubMed Central

    Lee, Dong-Ha; Kim, Hyun-Hong; Cho, Hyun-Jeong; Yu, Young-Bin; Kang, Hyo-Chan; Kim, Jong-Lae; Lee, Jong-Jin; Park, Hwa-Jin

    2014-01-01

    In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca2+]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca2+]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca2+]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca2+-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease. PMID:25009703

  19. SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

    PubMed

    Walsh, Tony G; Harper, Matthew T; Poole, Alastair W

    2015-01-01

    Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway.

  20. Role for the thromboxane A2 receptor β-isoform in the pathogenesis of intrauterine growth restriction

    PubMed Central

    Powell, Katie L.; Stevens, Veronica; Upton, Dannielle H.; McCracken, Sharon A.; Simpson, Ann M.; Cheng, Yan; Tasevski, Vitomir; Morris, Jonathan M.; Ashton, Anthony W.

    2016-01-01

    Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPβ) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPβ attenuated these functions and inhibited migration. Expression of the TPβ transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPβ impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans. PMID:27363493

  1. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  2. Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

    PubMed Central

    Ferré, Sergi; Quiroz, César; Orru, Marco; Guitart, Xavier; Navarro, Gemma; Cortés, Antonio; Casadó, Vicent; Canela, Enric I.; Lluis, Carme; Franco, Rafael

    2011-01-01

    A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders. PMID:21731559

  3. EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

    PubMed Central

    Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F.; Rudel, Thomas

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and

  4. The cyclooxygenase-2/thromboxane A2 pathway: a bridge from rheumatoid arthritis to lung cancer?

    PubMed

    Huang, Qing-Chun; Huang, Run-Yue

    2014-11-01

    Patients with rheumatoid arthritis (RA) appear to be at a higher risk of lung cancer (LC). Although the connection between RA and LC has been an active area of research for many years, the molecular pathogenesis of the disease process remains unclear. The cyclooxygenase (COX)-2/thromboxane A2 (TxA2) pathway has been shown to play a potential role in LC development through an auto-regulatory feedback loop. An increased level of TxA2 has been found in RA patients, and intriguingly, the positive feedback loop for the COX-2/TxA2 pathway was shown to have a potential function in RA fibroblast-like synoviocytes (RA-FLS). Thus, the molecular basis of LC development in patients with RA has been at least in partly described. It is possible that COX-2-derived TxA2 could be monitored for the early detection of LC in RA patients, and targeting this molecular pathway may decrease the risk of LC in patients with RA.

  5. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  6. Effects of heparin on platelet aggregation and release and thromboxane A2 production

    SciTech Connect

    Mohammad, S.F.; Anderson, W.H.; Smith, J.B.; Chuang, H.Y.; Mason, R.G.

    1981-08-01

    Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with /sup 3/H-arachidonic acid and /sup 14/C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of /sup 14/C-serotonin and /sup 3/H-arachidonic acid metabolites, it appeared that either release of /sup 14/C and /sup 3/H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both /sup 14/C and /sup 3/H.

  7. Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

    PubMed Central

    Guo, Dong; Mulder-Krieger, Thea; IJzerman, Adriaan P; Heitman, Laura H

    2012-01-01

    BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. PMID:22324512

  8. A2A Adenosine Receptor (A2AAR) as a Therapeutic Target in Diabetic Retinopathy

    PubMed Central

    Ibrahim, Ahmed S.; El-shishtawy, Mamdouh M.; Zhang, Wenbo; Caldwell, Ruth B.; Liou, Gregory I.

    2011-01-01

    In diabetic retinopathy (DR), abnormalities in vascular and neuronal function are closely related to the local production of inflammatory mediators whose potential source is microglia. A2A adenosine receptor (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in DR. Here, we evaluate the role of A2AAR and its underlying signaling in retinal complications associated with diabetes. Initial studies in wild-type mice revealed that the treatment with the A2AAR agonist resulted in marked decreases in hyperglycemia-induced retinal cell death and tumor necrosis factor (TNF)-α release. To further assess the role of A2AAR in DR, we studied the effects of A2AAR ablation on diabetes-induced retinal abnormalities. Diabetic A2AAR−/− mice had significantly more terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, TNF-α release, and intercellular adhesion molecule-1 expression compared with diabetic wild-type mice. To explore a potential mechanism by which A2AAR signaling regulates inflammation in DR, we performed additional studies using microglial cells treated with Amadori-glycated albumin, a risk factor in diabetic disorders. The results showed that activation of A2AAR attenuated Amadori-glycated albumin-induced TNF-α release in a cAMP/exchange protein directly activated by cAMP-dependent mechanism and significantly repressed the inflammatory cascade, C-Raf/extracellular signal-regulated kinase (ERK), in activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in DR and suggests that the retinal protective effect of A2AAR is mediated by abrogating the inflammatory response that occurs in microglia via interaction with C-Raf/ERK pathway. PMID:21514428

  9. Potential therapeutic relevance of adenosine A2B and A2A receptors in the central nervous system.

    PubMed

    Popoli, Patrizia; Pepponi, Rita

    2012-09-01

    Adenosine A2B and, much more importantly, adenosine A2A receptors modulate many physiological and pathological processes in the brain. In this review, the most recent evidence concerning the role of such receptors and their potential therapeutic relevance is discussed. The low affinity of A2B receptors for adenosine implies that they might represent a good therapeutic target, since they are activated only under pathological conditions (when adenosine levels raise up to micromolar concentrations). The availability of selective ligands for A2B receptors would allow exploration of such an hypothesis. Since adenosine A2A receptors mediate both potentially neuroprotective and potentially neurotoxic effects, their role in neurodegenerative diseases is highly controversial. Nevertheless, A2A receptor antagonists have shown clear antiparkinsonian effects, and a great interest exists on the role of A2A receptors in Alzheimer's disease, brain ischaemia, spinal cord injury, drug addiction and other conditions. In order to establish whether such receptors represent a target for CNS diseases, at least two conditions are needed: the full comprehension of A2A-dependent mechanisms and the availability of ligands capable of discriminating among the different receptor populations.

  10. Remodeling of striatal NMDA receptors by chronic A(2A) receptor blockade in Huntington's disease mice.

    PubMed

    Martire, Alberto; Ferrante, Antonella; Potenza, Rosa Luisa; Armida, Monica; Ferretti, Roberta; Pézzola, Antonella; Domenici, Maria Rosaria; Popoli, Patrizia

    2010-01-01

    Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A(2A)R blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A(2A)R blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A(2A)Rs in HD.

  11. Adenosine receptors and diabetes: Focus on the A(2B) adenosine receptor subtype.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Gessi, Stefania

    2015-09-01

    Over the last two decades, diabetes mellitus has become one of the most challenging health problems worldwide. Diabetes mellitus, classified as type I and II, is a pathology concerning blood glucose level in the body. The nucleoside adenosine has long been known to affect insulin secretion, glucose homeostasis and lipid metabolism, through activation of four G protein coupled adenosine receptors (ARs), named A1, A2A, A2B and A3. Currently, the novel promising subtype to develop new drugs for diabetes treatment is the A2BAR subtype. The use of selective agonists and antagonists for A2BAR subtype in various diabetic animal models allowed us to identify several effects of A2BAR signaling in cell metabolism. In particular, the focus of this review is to summarize the studies on purinergic signaling associated with diabetes through A2BARs modulation.

  12. TP receptor as a therapeutic target in atherosclerosis and related cardiovascular diseases.

    PubMed

    Ishizuka, Toshiaki; Higashino, Hideaki

    2006-11-01

    It had already showed that several thromboxane A(2) receptor (TP receptor) antagonists might be utilized in the treatment of cardiovascular diseases. In addition, recent reports suggested that TP receptor antagonism may be able to restrict vascular inflammation in atherosclerotic vessels. In particular, S18886 has been developed as a non-prostanoid TP receptor antagonist derived from sulotroban that is characterized by a tetrahydronaphthalene ring as a spacer between the 4-cholophenylsulfonamide group and carboxylic acid. Several reports using experimental animal models of atherosclerosis indicated that S18886 caused a regression of advanced atherosclerosis. More recently, several studies and patents showed that new thromboxane modulators combined with another pharmacological activities have been developed. Ohtake et al. discovered TRA-418 (a benzene-condensed heterocyclic derivative) having a TP receptor antagonistic activity and a prostaglandin I(2) receptor agonistic activity. Cesagrande found that 4-methyl-N- (4-trans-nitrooxycyclohexyl)-N-(3-pyridinylmethyl)-1,3- benzenedicarboxamide is endowed with anti-thromboxane and NO-donor actions. Oketani et al. discovered that E3040, a novel benzothiazole derivative, inhibited TXA(2) synthase and 5-LO activities. EK112, a combined angiotensin II and TP receptor antagonist, was developed. These new compounds may be able to restrict further infiltration of inflammatory cells in atherosclerotic vessels, thus stabilizing vulnerable plaques in the related cardiovascular diseases.

  13. A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

    PubMed

    Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

    2017-01-01

    Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders.

  14. Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets

    PubMed Central

    Moraes, Leonardo A.; Swales, Karen E.; Wray, Jessica A.; Damazo, Amilcar; Gibbins, Jonathan M.; Warner, Timothy D.

    2007-01-01

    Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) γ, PPARβ, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRα and RXRβ. RXR ligands inhibit platelet aggregation and TXA2 release to ADP and the TXA2 receptors, but only weakly to collagen. ADP and TXA2 both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families. PMID:17213293

  15. Role of adenosine A2b receptor overexpression in tumor progression.

    PubMed

    Sepúlveda, Cesar; Palomo, Iván; Fuentes, Eduardo

    2016-12-01

    The adenosine A2b receptor is a G-protein coupled receptor. Its activation occurs with high extracellular adenosine concentration, for example in inflammation or hypoxia. These conditions are generated in the tumor environment. Studies show that A2b receptor is overexpressed in various tumor lines and biopsies from patients with different cancers. This suggests that A2b receptor can be used by tumor cells to promote progression. Thus A2b participates in different events, such as angiogenesis and metastasis, besides exerting immunomodulatory effects that protect tumor cells. Therefore, adenosine A2b receptor appears as an interesting therapeutic target for cancer treatment.

  16. Potential therapeutic interest of adenosine A2A receptors in psychiatric disorders.

    PubMed

    Cunha, Rodrigo A; Ferré, Sergi; Vaugeois, Jean-Marie; Chen, Jiang-Fan

    2008-01-01

    The interest on targeting adenosine A(2A) receptors in the realm of psychiatric diseases first arose based on their tight physical and functional interaction with dopamine D(2) receptors. However, the role of central A(2A) receptors is now viewed as much broader than just controlling D(2) receptor function. Thus, there is currently a major interest in the ability of A(2A) receptors to control synaptic plasticity at glutamatergic synapses. This is due to a combined ability of A(2A) receptors to facilitate the release of glutamate and the activation of NMDA receptors. Therefore, A(2A) receptors are now conceived as a normalizing device promoting adequate adaptive responses in neuronal circuits, a role similar to that fulfilled, in essence, by dopamine. This makes A(2A) receptors particularly attractive targets to manage psychiatric disorders since adenosine may act as go-between glutamate and dopamine, two of the key players in mood processing. Furthermore, A(2A) receptors also control glia function and brain metabolic adaptation, two other emerging mechanisms to understand abnormal processing of mood, and A(2A) receptors are important players in controlling the demise of neurodegeneration, considered an amplificatory loop in psychiatric disorders. Current data only provide an indirect confirmation of this putative role of A(2A) receptors, based on the effects of caffeine (an antagonist of both A(1) and A(2A) receptors) in psychiatric disorders. However, the introduction of A(2A) receptors antagonists in clinics as anti-parkinsonian agents is hoped to bolster our knowledge on the role of A(2A) receptors in mood disorders in the near future.

  17. The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction.

    PubMed

    Filip, M; Zaniewska, M; Frankowska, M; Wydra, K; Fuxe, K

    2012-01-01

    Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies

  18. Adenosine A2A receptor antagonism and neuroprotection: mechanisms, lights, and shadows.

    PubMed

    Popoli, Patrizia; Minghetti, Luisa; Tebano, Maria Teresa; Pintor, Annita; Domenici, Maria Rosaria; Massotti, Marino

    2004-01-01

    Adenosine A2A receptor antagonists are regarded as potential neuroprotective drugs, although the mechanisms underlying their effects remain to be elucidated. In this review, quinolinic acid (QA)-induced striatal toxicity was used as a tool to investigate the mechanisms of the neuroprotective effects of A2A receptor antagonists. After having examined the effects of selective A2A receptor antagonists toward different mechanisms of QA toxicity, we conclude that (1) the effect elicited by A2A receptor blockade on QA-induced glutamate outflow may be one of the mechanisms of the neuroprotective activity of A2A receptor antagonists; (2) A2A receptor antagonists have a potentially worsening influence on QA-dependent NMDA receptor activation; and (3) the ability of A2A receptor antagonists to prevent QA-induced lipid peroxidation does not correlate with the neuroprotective effects. These results suggest that A2A receptor antagonists may have either potentially beneficial or detrimental influence in models of neurodegeneration that are mainly due to increased glutamate levels or enhanced sensitivity of NMDA receptors, respectively.

  19. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  20. Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors.

    PubMed

    Ferré, Sergi; Sebastião, Ana Maria

    2016-03-01

    This Editorial highlights a study by Chiodi et al. () showing that the effects mediated by cannabinoid CB1 receptor (CB1R) activation in the striatum are significantly reduced in rats with neuronal over-expression of adenosine A2A receptors (A2AR). Two hypotheses are derived from that study. Hypothesis A: two subpopulations of pre-synaptic CB1R in corticostriatal glutamatergic terminals exist, one forming and another not forming heteromers with A2AR. Hypothesis B: CB1R are predominantly forming heteromers with A2AR. In the case of hypothesis A, the A2AR might be required for CB1R-A2AR heteromeric signaling, whereas non-heteromeric CB1R activity is inhibited by A2ARs. In the case of hypothesis B, up-regulation of A2ARs may perturb heteromeric stoichiometry, thus reducing CB1R functioning. In any case, pre-synaptic striatal A2AR-CB1R heteromers emerge as important targets of the effects of cannabinoids demonstrated at the neuronal and behavioral level. Read the highlighted article 'Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors' on page 907.

  1. Adenosine A2B Receptor: From Cell Biology to Human Diseases

    PubMed Central

    Sun, Ying; Huang, Pingbo

    2016-01-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR's functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases, and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases. PMID:27606311

  2. Adenosine A2B receptor: from cell biology to human diseases

    NASA Astrophysics Data System (ADS)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

  3. A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

    PubMed Central

    Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

    2012-01-01

    In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

  4. A critical evaluation of adenosine A2A receptors as potentially "druggable" targets in Huntington's disease.

    PubMed

    Popoli, Patrizia; Blum, David; Domenici, Maria Rosaria; Burnouf, Sylvie; Chern, Yijuang

    2008-01-01

    Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the expansion of a polymorphic CAG trinucleotide repeat encoding a poly-glutamine tract within the Huntingtin protein. GABAergic enkephalin neurons of the basal ganglia, which show the highest levels of expression of adenosine A(2A) receptors, are the most vulnerable in HD. Such a selective neuronal vulnerability, which occurs despite ubiquitous expression of mutant and normal Huntingtin, has suggested that adenosine A(2A) receptors might play a pathogenetic role in HD. In agreement, changes in A(2A) receptor expression and signaling have been reported in various experimental models of HD. The interpretation of the functional significance of the aberrant A(2A) receptor phenotype in HD mice is however complicated by the conflicting data so far reported on the potential neuroprotective and neurodegenerative effects of these receptors in the brain, with some data suggesting a potential pathogenetic role and some other data suggesting activation of trophic or protective pathways in neurons. The same complex profile has emerged in experimental models of HD, in which both A(2A) receptor agonists and antagonists have shown beneficial effects. The main aim of this review is to critically evaluate whether adenosine A(2A) receptors may represent a suitable target to develop drugs against HD.

  5. GABAergic involvement in motor effects of an adenosine A(2A) receptor agonist in mice.

    PubMed

    Khisti, R T; Chopde, C T; Abraham, E

    2000-04-03

    Adenosine A(2A) agonists are known to induce catalepsy and inhibit dopamine mediated motor hyperactivity. An antagonistic interaction between adenosine A(2A) and dopamine D(2) receptors is known to regulate GABA-mediated neurotransmission in striatopallidal neurons. Stimulation of adenosine A(2A) and dopamine D(2) receptors has been shown to increase and inhibit GABA release respectively in pallidal GABAergic neurons. However, the role of GABAergic neurotransmission in the motor effects of adenosine A(2A) receptors is not yet known. Therefore in the present study the effect of GABAergic agents on adenosine A(2A) receptor agonist (NECA- or CGS 21680) induced catalepsy and inhibition of amphetamine elicited motor hyperactivity was examined. Pretreatment with GABA, the GABA(A) agonist muscimol or the GABA(B) agonist baclofen potentiated whereas the GABA(A) antagonist bicuculline attenuated NECA- or CGS 21680-induced catalepsy. However, the GABA(B) antagonists phaclophen and delta-aminovaleric acid had no effect. Administration of NECA or CGS 21680 not only reduced spontaneous locomotor activity but also antagonized amphetamine elicited motor hyperactivity. These effects of NECA and CGS 21680 were potentiated by GABA or muscimol and antagonized by bicuculline. These findings provide behavioral evidence for the role of GABA in the motor effects of adenosine A(2A) receptor agonists. Activation of adenosine A(2A) receptors increases GABA release which could reduce dopaminergic tone and induce catalepsy or inhibit amphetamine mediated motor hyperactivity.

  6. Adenosine A2 receptors modulate haloperidol-induced catalepsy in rats.

    PubMed

    Mandhane, S N; Chopde, C T; Ghosh, A K

    1997-06-11

    The effect of adenosine A1 and A2 receptor agonists and antagonists was investigated on haloperidol-induced catalepsy in rats. Pretreatment (i.p.) with the non-selective adenosine receptor antagonist, theophylline, or the selective adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), significantly reversed haloperidol-induced catalepsy, whereas the selective adenosine A1 receptor antagonists, 8-phenyltheophylline and 8-cyclopentyl-1,3-dipropylxanthine produced no effect. Similar administration of the adenosine A2 receptor agonists, 5'-(N-cyclopropyl)-carboxamidoadenosine and 5'-N-ethylcarboxamidoadenosine (NECA), and the mixed agonists with predominantly A1 site of action, N6-(2-phenylisopropyl) adenosine or 2-chloroadenosine, potentiated haloperidol-induced catalepsy. Higher doses of the adenosine agonists produced catalepsy when given alone. However, N6-cyclopentyladenosine, a highly selective adenosine A1 receptor agonist, was ineffective in these respects. The per se cataleptic effect of adenosine agonists was blocked by DMPX and the centrally acting anticholinergic agent, scopolamine. Scopolamine also attenuated the potentiation of haloperidol-induced catalepsy by adenosine agonists. Further, i.c.v. administration of NECA and DMPX produced a similar effect as that produced after their systemic administration. These findings demonstrate the differential influence of adenosine A1 and A2 receptors on haloperidol-induced catalepsy and support the hypothesis that the functional interaction between adenosine and dopamine mechanisms might occur through adenosine A2 receptors at the level of cholinergic neurons. The results suggest that adenosine A2, but not A1, receptor antagonists may be of potential use in the treatment of Parkinson's disease.

  7. The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity

    PubMed Central

    Koupenova, Milka; Carroll, Shannon; Ravid, Katya

    2014-01-01

    High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR null background. Reinstatement of macrophage A2bAR expression in A2bAR null mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice. PMID:24892847

  8. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    PubMed Central

    Pedata, Felicita; Pugliese, Anna Maria; Coppi, Elisabetta; Dettori, Ilaria; Maraula, Giovanna; Cellai, Lucrezia; Melani, Alessia

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke. PMID:25165414

  9. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  10. A2a and a2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Stefanelli, Angela; Bencivenni, Serena; Castillo, Carlos Alberto; Varani, Katia; Gessi, Stefania

    2015-05-15

    Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A2A through p44/42 and A2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. GLIA 2015.

  11. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    PubMed

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  12. Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection

    PubMed Central

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    G protein coupled receptors and tropomyosin-related kinase (Trk) receptors have distinct structure and transducing mechanisms; therefore, cross-talk among them was unexpected. Evidence has, however, accumulated showing that tonic adenosine A2A receptor activity is a required step to allow synaptic actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. An enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor in aged animals, which might prove particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as glial derived neurotrophic factor at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurones will be reviewed and discussed in the light of therapeutic strategies for neurodegenerative diseases. PMID:19508402

  13. A role of the SAM domain in EphA2 receptor activation

    PubMed Central

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W.

    2017-01-01

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization. PMID:28338017

  14. A2A Adenosine Receptor Antagonism Enhances Synaptic and Motor Effects of Cocaine via CB1 Cannabinoid Receptor Activation

    PubMed Central

    Tozzi, Alessandro; de Iure, Antonio; Marsili, Valentina; Romano, Rosaria; Tantucci, Michela; Di Filippo, Massimiliano; Costa, Cinzia; Napolitano, Francesco; Mercuri, Nicola Biagio; Borsini, Franco; Giampà, Carmen; Fusco, Francesca Romana; Picconi, Barbara; Usiello, Alessandro; Calabresi, Paolo

    2012-01-01

    Background Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. Principal Findings Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. Conclusions The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine. PMID:22715379

  15. Therapeutic Opportunities for Caffeine and A2A Receptor Antagonists in Retinal Diseases.

    PubMed

    Boia, Raquel; Ambrósio, António Francisco; Santiago, Ana Raquel

    2016-01-01

    Caffeine, the major component of coffee, is the most consumed psychostimulant in the world. Caffeine is an adenosine analog and acts as a nonselective adenosine receptor antagonist. The majority of the effects of caffeine are mainly mediated by the blockade of adenosine receptors, and the proved neuroprotective effects of caffeine in brain disorders have been mimicked by the blockade of adenosine A2A receptor (A2AR). A growing body of evidence demonstrates that microglia-mediated neuroinflammation plays a key role in the pathophysiology of brain and retinal diseases. Moreover, the control of microglia reactivity by blocking A2AR has been proposed to be the mechanism underlying the observed protective effects of caffeine. Hence, it is conceivable that caffeine and A2AR antagonists offer therapeutic value for the treatment of retinal diseases, mainly those involving microglia-mediated neuroinflammation.

  16. Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

    PubMed Central

    Pagnussat, N; Almeida, A S; Marques, D M; Nunes, F; Chenet, G C; Botton, P H S; Mioranzza, S; Loss, C M; Cunha, R A; Porciúncula, L O

    2015-01-01

    Background and Purpose Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer’s disease, an effect mimicked by adenosine A2A receptor, but not A1 receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice. Experimental Approach We determined whether A2A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. Key Results Scopolamine (1.0 mg·kg−1, i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A2A receptor antagonist (SCH 58261, 0.1–1.0 mg·kg−1, i.p.) and by the A1 receptor antagonist (DPCPX, 0.2–5.0 mg·kg−1, i.p.), except in the modified Y-maze where only SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A2A receptors with CGS 21680 (0.1–0.5 mg·kg−1, i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg−1, i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. Conclusions and Implications These results show that A2A receptors are necessary and sufficient to trigger memory impairment and further suggest that A1 receptors might also be selectively engaged to control the cholinergic-driven memory impairment. PMID:25939452

  17. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons.

    PubMed

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L; Anggono, Victor; Gether, Ulrik; Huganir, Richard L; Madsen, Kenneth L

    2013-08-27

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na(+)/H(+)-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.

  18. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells.

    PubMed

    Wilson, Jeffrey M; Ross, William G; Agbai, Oma N; Frazier, Renea; Figler, Robert A; Rieger, Jayson; Linden, Joel; Ernst, Peter B

    2009-04-15

    The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

  19. Targeting Adenosine A2A Receptors in Parkinson’s Disease

    DTIC Science & Technology

    2006-11-01

    dysfunction in the rat hippocampus (collaboration with MA Lynch). Conclusion: These results prompt the hypothesis that the neuroprotection associated...stimuli and show reduced responses to morphine . The role of the A2A receptor in pain is less clear and we, and others, have shown the receptor is absent...cerebral cortex, striatum, hippocampus , olfactory bulb, and hypothalamus) but not in the cerebellum. (2), immunohistochemical analysis showed that

  20. The A2a adenosine receptor modulates the reinforcement efficacy and neurotoxicity of MDMA.

    PubMed

    Ruiz-Medina, Jessica; Ledent, Catherine; Carretón, Olga; Valverde, Olga

    2011-04-01

    Adenosine is an endogenous purine nucleoside that plays a neuromodulatory role in the central nervous system. A2a adenosine receptors have been involved in reward-related processes, inflammatory phenomena and neurotoxicity reactions. In the present study, we investigated the role of A2a adenosine receptors on the acute pharmacological effects, reinforcement and neuroinflammation induced by MDMA administration. First, the acute effects of MDMA on body temperature, locomotor activity and anxiety-like responses were measured in A2a knockout mice and wild-type littermates. Second, MDMA reinforcing properties were evaluated using the intravenous self-administration paradigm. Finally, we assessed striatal astrogliosis and microgliosis as markers of MDMA neurotoxicity. Our results showed that acute MDMA produced a biphasic effect on body temperature and increased locomotor activity and anxiogenic-like responses in both genotypes. However, MDMA reinforcing properties were dramatically affected by the lack of A2a adenosine receptors. Thus, wild-type mice maintained MDMA self-administration under a fixed ratio 1 reinforcement schedule, whereas the operant response appeared completely abolished in A2a knockout mice. In addition, the MDMA neurotoxic regime produced an enhanced inflammatory response in striatum of wild-type mice, revealed by a significant increase in glial expression, whereas such activation was attenuated in mutant mice. This is the first report indicating that A2a adenosine receptors play a key role in reinforcement and neuroinflammation induced by the widely used psychostimulant.

  1. Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

    PubMed

    Brozmanova, M; Mazurova, L; Ru, F; Tatar, M; Hu, Y; Yu, S; Kollarik, M

    2016-02-01

    Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

  2. Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Chiodi, Valentina; Pepponi, Rita; Ferrante, Antonella; Domenici, Maria Rosaria; Frank, Claudio; Chen, Jiang-Fan; Ledent, Catherine; Popoli, Patrizia

    2009-09-01

    Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.

  3. The impact of adenosine and A(2B) receptors on glucose homoeostasis.

    PubMed

    Rüsing, D; Müller, C E; Verspohl, E J

    2006-12-01

    Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

  4. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

    PubMed Central

    Yang, Dan; Zhang, Ying; Nguyen, Hao G.; Koupenova, Milka; Chauhan, Anil K.; Makitalo, Maria; Jones, Matthew R.; Hilaire, Cynthia St.; Seldin, David C.; Toselli, Paul; Lamperti, Edward; Schreiber, Barbara M.; Gavras, Haralambos; Wagner, Denisa D.; Ravid, Katya

    2006-01-01

    Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor–knockout/reporter gene–knock-in (A2BAR-knockout/reporter gene–knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-α, and a consequent downregulation of IκB-α are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target. PMID:16823489

  5. Silencing Receptor EphA2 Enhanced Sensitivity to Lipoplatin™ in Lung Tumor and MPM Cells.

    PubMed

    Lee, Hung-Yen; Mohammed, Kamal A; Goldberg, Eugene P; Kaye, Frederic; Najmunnisa, Nasreen

    2016-08-08

    Receptor EphA2 is overexpressed in lung cancer and malignant pleural mesothelioma (MPM) which promote tumorogenesis. Lipoplatin™, a new liposomal cisplatin formulation, is used against resistant tumors. Use of cisplatin-based drugs leads to unacceptable toxicities. To improve the effectiveness of Lipoplatin, enhancing the cellular sensitivity of lung tumor and MPM cells is critical. Therefore, we targeted receptor EphA2 by silencing interference RNA (siRNA) and treated tumor cells with Lipoplatin. The combined effects of siRNA-EphA2 and Lipoplatin were determined. We report that silencing EphA2 significantly enhanced the cellular sensitivity of lung tumor and MPM cells to Lipoplatin and maybe a potential therapy for lung cancer.

  6. A2A adenosine receptors are up-regulated in lymphocytes from amyotrophic lateral sclerosis patients.

    PubMed

    Vincenzi, Fabrizio; Corciulo, Carmen; Targa, Martina; Casetta, Ilaria; Gentile, Mauro; Granieri, Enrico; Borea, Pier Andrea; Popoli, Patrizia; Varani, Katia

    2013-09-01

    Adenosine, a purine nucleoside interacting with A1, A2A, A2B and A3 adenosine receptors (ARs), is a potent endogenous modulator of inflammatory and neuronal processes involved in the pathophysiology of several neurodegenerative diseases. In the present study, ARs were investigated in lymphocytes from patients with amyotrophic lateral sclerosis (ALS) and compared with age-matched healthy subjects. In ALS patients A2AARs were analysed by using RT-PCR, Western blotting and saturation binding experiments. The effect of A2AAR stimulation on cyclic AMP levels was evaluated in lymphocytes from ALS patients and healthy subjects. An up-regulation of A2AARs was observed in ALS patients with respect to healthy subjects while A1, A2B and A3AR affinity and density did not change. In ALS patients, the A2AAR density values correlated with the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) scores. Furthermore, the stimulation of A2AARs mediated a significant increase in cyclic AMP levels in lymphocytes from ALS patients, with a higher potency than in lymphocytes from healthy subjects. In conclusion, the positive correlation between A2AAR density and ALSFRS-R scores could indicate a possible protective effect of this receptor subtype, representing an interesting starting point for the study of alternative therapeutic approaches for ALS based on A2AAR modulation.

  7. Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

    PubMed Central

    Ledent, C; Dumont, J E; Vassart, G; Parmentier, M

    1992-01-01

    Cyclic AMP (cAMP) is the major intracellular second messenger of thyrotropin (TSH) action on thyroid cells. It stimulates growth as well as the function and differentiation of cultured thyrocytes. The adenosine A2 receptor, which activates adenylyl cyclase via coupling to the stimulating G protein (Gs), has been shown to promote constitutive activation of the cAMP cascade when transfected into various cell types. In order to test whether the A2 receptor was able to function similarly in vivo and to investigate the possible consequences of permanent adenylyl cyclase activation in thyroid cells, lines of transgenic mice were generated expressing the canine A2 adenosine receptor under control of the bovine thyroglobulin gene promoter. Thyroid-specific expression of the A2 adenosine receptor transgene promoted gland hyperplasia and severe hyperthyroidism causing premature death of the animals. The resulting goitre represents a model of hyperfunctioning adenomas: it demonstrates that constitutive activation of the cAMP cascade in such differentiated epithelial cells is sufficient to stimulate autonomous and uncontrolled function and growth. Images PMID:1371462

  8. Multiple sclerosis lymphocytes upregulate A2A adenosine receptors that are antiinflammatory when stimulated.

    PubMed

    Vincenzi, Fabrizio; Corciulo, Carmen; Targa, Martina; Merighi, Stefania; Gessi, Stefania; Casetta, Ilaria; Gentile, Mauro; Granieri, Enrico; Borea, Pier Andrea; Varani, Katia

    2013-08-01

    Multiple sclerosis (MS) is an autoimmune-mediated inflammatory disease characterized by multifocal areas of demyelination. Experimental evidence indicates that A2A adenosine receptors (ARs) play a pivotal role in the inhibition of inflammatory processes. The aim of this study was to investigate the contribution of A2A ARs in the inhibition of key pro-inflammatory mediators for the pathogenesis of MS. In lymphocytes from MS patients, A1, A2A, A2B, and A3 ARs were analyzed by using RT-PCR, Western blotting, immunofluorescence, and binding assays. Moreover the effect of A2A AR stimulation on proinflammatory cytokine release such as TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and on lymphocyte proliferation was evaluated. The capability of an A2A AR agonist on the modulation of very late antigen (VLA)-4 expression and NF-κB was also explored. A2A AR upregulation was observed in lymphocytes from MS patients in comparison with healthy subjects. The stimulation of these receptors mediated a significant inhibition of TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and cell proliferation as well as VLA-4 expression and NF-κB activation. This new evidence highlights that A2A AR agonists could represent a novel therapeutic tool for MS treatment as suggested by the antiinflammatory role of A2A ARs in lymphocytes from MS patients.

  9. High salt diet exacerbates vascular contraction in the absence of adenosine A2A receptor

    PubMed Central

    Pradhan, Isha; Zeldin, Darryl C.; Ledent, Catherine; Mustafa, S. Jamal; Falck, John R.; Nayeem, Mohammed A

    2014-01-01

    High salt (4%NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A2A-receptor (A2AAR). Evidence suggests A2AAR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A2AAR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A2AAR+/+, but exaggerates contraction in A2AAR−/−. Organ-bath and Western-blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A2AAR+/+ and A2AAR−/− mice aortae. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A2AAR+/+, whereas contraction was observed in A2AAR−/− mice and this was attenuated by A1AR antagonist (DPCPX). CGS-21680 (selective A2AAR-agonist) enhanced relaxation in HS-A2AAR+/+ vs. NS-A2AAR+/+, that was blocked by EETs antagonist (14,15-EEZE). Compared to NS, HS significantly upregulated expression of vasodilators A2AAR and cyp2c29, while vasoconstrictors A1AR and cyp4a in A2AAR+/+ were downregulated. In A2AAR−/− mice, however, HS significantly downregulated the expression of cyp2c29, while A1AR and cyp4a were upregulated compared to A2AAR+/+ mice. Hence, our data suggest that in A2AAR+/+, HS enhances A2AAR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A1AR levels, whereas in A2AAR−/−, HS exaggerates contraction through decreased cyp-epoxygenases and increased A1AR levels. PMID:24390173

  10. Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2

    SciTech Connect

    Salaita, Khalid; Nair, Pradeep M; Petit, Rebecca S; Neve, Richard M; Das, Debopriya; Gray, Joe W; Groves, Jay T

    2009-09-09

    Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.

  11. A covalent antagonist for the human adenosine A2A receptor.

    PubMed

    Yang, Xue; Dong, Guo; Michiels, Thomas J M; Lenselink, Eelke B; Heitman, Laura; Louvel, Julien; IJzerman, Ad P

    2016-12-03

    The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.

  12. Functions, dysfunctions and possible therapeutic relevance of adenosine A2A receptors in Huntington's disease.

    PubMed

    Popoli, Patrizia; Blum, David; Martire, Alberto; Ledent, Catherine; Ceruti, Stefania; Abbracchio, Maria P

    2007-04-01

    The aim of this review is to summarize and critically discuss the complex role played by adenosine A(2A) receptors (A(2A)Rs) in Huntington's disease (HD). Since A(2A)Rs are mainly localized on the neurons, which degenerate early in HD, and given their ability to stimulate glutamate outflow and inflammatory gliosis, it was hypothesized that they could be involved in the pathogenesis of HD, and that A(2A)R antagonists could be neuroprotective. This was further sustained by the demonstration that A(2A)Rs and underlying signaling systems undergo profound changes in cellular and animal models of HD. More recently, however, the equation A(2A) receptor blockade=neuroprotection has appeared too simplistic. First, it is now definitely clear that, besides mediating 'bad' responses (for example, stimulation of glutamate outflow and excessive glial activation), A(2A)Rs also promote 'good' responses (such as trophic and antinflammatory effects). This implies that A(2A)R blockade results either in pro-toxic or neuroprotective effects according to the mechanisms involved in a given experimental model. Second, since HD is a chronically progressive disease, the multiple mechanisms involving A(2A)Rs may play different relative roles along the degenerative process. Such different mechanisms can be influenced by A(2A)R activation or blockade in different ways, even leading to opposite outcomes depending on the time of agonist/antagonist administration. The number, and the complexity, of the possible scenarios is further increased by the influence of mutant Huntingtin on both the expression and functions of A(2A)Rs, and by the strikingly different effects mediated by A(2A)Rs expressed by different cell populations within the brain.

  13. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease

    PubMed Central

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D.; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K.; Blackwell, Timothy S.; Xia, Yang; Johnston, Richard A.; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R.

    2012-01-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A2BR or treatment with the A2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.—Karmouty-Quintana, H., Zhong, H., Acero, L., Weng, T., Melicoff, E., West, J. D., Hemnes, A., Grenz, A., Eltzschig, H. K., Blackwell, T. S., Xia, Y., Johnston, R. A., Zeng, D., Belardinelli, L., Blackburn, M. R. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease. PMID:22415303

  14. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor

  15. An update on non-peptide angiotensin receptor antagonists and related RAAS modulators.

    PubMed

    Aulakh, G K; Sodhi, R K; Singh, M

    2007-08-02

    The renin-angiotensin-aldosterone-system (RAAS) is an important regulator of blood pressure and fluid-electrolyte homeostasis. RAAS has been implicated in pathogenesis of hypertension, congestive heart failure, and chronic renal failure. Aliskiren is the first non-peptide orally active renin inhibitor approved by FDA. Angiotensin Converting Enzyme (ACE) Inhibitors are associated with frequent side effects such as cough and angio-oedema. Recently, the role of ACE2 and neutral endopeptidase (NEP) in the formation of an important active metabolite/mediator of RAAS, ang 1-7, has initiated attempts towards development of ACE2 inhibitors and combined ACE/NEP inhibitors. Furukawa and colleagues developed a series of low molecular weight nonpeptide imidazole analogues that possess weak but selective, competitive AT1 receptor blocking property. Till date, many compounds have exhibited promising AT1 blocking activity which cause a more complete RAAS blockade than ACE inhibitors. Many have reached the market for alternative treatment of hypertension, heart failure and diabetic nephropathy in ACE inhibitor intolerant patients and still more are waiting in the queue. But, the hallmark of this area of drug research is marked by a progress in understanding molecular interaction of these blockers at the AT1 receptor and unraveling the enigmatic influence of AT2 receptors on growth/anti-growth, differentiation and the regeneration of neuronal tissue. Different modeling strategies are underway to develop tailor made molecules with the best of properties like Dual Action (Angiotensin And Endothelin) Receptor Antagonists (DARA), ACE/NEP inhibitors, triple inhibitors, AT2 agonists, AT1/TxA2 antagonists, balanced AT1/AT2 antagonists, and nonpeptide renin inhibitors. This abstract gives an overview of these various angiotensin receptor antagonists.

  16. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies

    DTIC Science & Technology

    2012-12-01

    in Traumatic Optic Neuropathies ” PRINCIPAL INVESTIGATOR: Gregory I. Liou, PhD CONTRACTING ORGANIZATION: Georgia Health Sciences...Adenosine Receptor A2A in Traumatic Optic Neuropathies 5b. GRANT NUMBER W81XWH-11-2-0046 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...ABSTRACT Our goal is to develop an early therapeutic intervention before the progression of traumatic optic neuropathy (TON), a vision-threatening

  17. Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

    PubMed

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N; Borycz, Janusz; Kachroo, Anil; Canas, Paula M; Orru, Marco; Schwarzschild, Michael A; Rosin, Diane L; Kreitzer, Anatol C; Cunha, Rodrigo A; Watanabe, Masahiko; Ferré, Sergi

    2009-11-18

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

  18. Reduced striatal adenosine A2A receptor levels define a molecular subgroup in schizophrenia.

    PubMed

    Villar-Menéndez, Izaskun; Díaz-Sánchez, Sara; Blanch, Marta; Albasanz, José Luis; Pereira-Veiga, Thais; Monje, Alfonso; Planchat, Luis Maria; Ferrer, Isidre; Martín, Mairena; Barrachina, Marta

    2014-04-01

    Schizophrenia (SZ) is a mental disorder of unknown origin. Some scientific evidence seems to indicate that SZ is not a single disease entity, since there are patient groups with clear symptomatic, course and biomarker differences. SZ is characterized by a hyperdopaminergic state related to high dopamine D2 receptor activity. It has also been proposed that there is a hypoadenosynergic state. Adenosine is a nucleoside widely distributed in the organism with neuromodulative and neuroprotective activity in the central nervous system. In the brain, the most abundant adenosine receptors are A1R and A2AR. In the present report, we characterize the presence of both receptors in human postmortem putamens of patients suffering SZ with real time TaqMan PCR, western blotting and radioligand binding assay. We show that A1R levels remain unchanged with respect to age-matched controls, whereas nearly fifty percent of patients have reduced A2AR, at the transcriptional and translational levels. Moreover, we describe how DNA methylation plays a role in the pathological A2AR levels with the bisulfite-sequencing technique. In fact, an increase in 5-methylcytosine percentage in the 5' UTR region of ADORA2A was found in those SZ patients with reduced A2AR levels. Interestingly, there was a relationship between the A2A/β-actin ratio and motor disturbances as assessed with some items of the PANSS, AIMS and SAS scales. Therefore, there may be a subgroup of SZ patients with reduced striatal A2AR levels accompanied by an altered motor phenotype.

  19. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    DTIC Science & Technology

    2016-03-01

    diabetic retinopathy. Life Sci. 2013 Jul 30;93(2-3):78-88. doi: 10.1016/j.lfs.2013.05.024. Epub 2013 Jun 12.PMID:23770229 7 AIMS: This study was...undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling...via A2A receptors (A2AAR) is involved in retinal protection from diabetes -induced inflammation. Here we demonstrate that AKI-enhanced adenosine

  20. Clinical/pharmacological aspect of adenosine A2A receptor antagonist for dyskinesia.

    PubMed

    Kanda, Tomoyuki; Uchida, Shin-ichi

    2014-01-01

    Dopamine replacement therapy using the dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), with a peripheral dopa decarboxylase inhibitor is the most effective treatment currently available for the symptoms of Parkinson's disease (PD). However, the long-term use of dopaminergic therapies for PD is often limited by the development of motor response complications, such as dyskinesia. Adenosine A2A receptors are a promising nondopaminergic target for the treatment of PD. The treatment of motor response complications involves combinations of regular and controlled release L-DOPA, perhaps with the addition of a COMT inhibitor or the use of a longer-acting dopamine agonist. However, when dyskinesia is already established, the increase in dopaminergic load produced by the addition of a dopamine agonist can result in an increase in the severity and duration of dyskinesia. Currently, there are no well-tolerated antidyskinesia agents available. Amantadine, which may exert its effects through the inhibition of N-methyl-D-aspartate (NMDA) receptors, shows some effects on established dyskinesia. Dyskinesia has a negative impact on the quality of life of patients, sometimes being more disabling than PD itself. Although some patients prefer experiencing dyskinesia than being in the OFF state and unable to move, alternative, more effective therapies are still required for severe disabling dyskinesia to afford patients an improved quality of life while in the ON state. The mechanisms causing and maintaining the dyskinesia have not been clarified. The application of a nondopaminergic approach to modify the basal ganglial activity would be helpful to better understand and treat dyskinesia. The use of an adenosine A2A receptor may provide one such approach. In this literature review, we will summarize the current knowledge from both clinical and nonclinical studies on the effects of adenosine A2A receptor blockade on dyskinesia.

  1. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    PubMed

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands.

  2. Molecular Determinants of CGS21680 Binding to the Human Adenosine A2A Receptor.

    PubMed

    Lebon, Guillaume; Edwards, Patricia C; Leslie, Andrew G W; Tate, Christopher G

    2015-06-01

    The adenosine A2A receptor (A(2A)R) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5'-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A(2A)R structures bound to either CGS21680, 5'-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169(EL2), His264(EL3), Leu267(7.32), and Ile274(7.39), and the amine group forms a hydrogen bond with the side chain of Ser67(2.65). Of these residues, only Ile274(7.39) is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A(2A)R bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser67(2.65) to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser67(2.65), Glu169(EL2), and His264(EL3), and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A(2A)R.

  3. Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils

    PubMed Central

    Varani, Katia; Gessi, Stefania; Merighi, Stefania; Iannotta, Valeria; Cattabriga, Elena; Spisani, Susanna; Cadossi, Ruggero; Borea, Pier Andrea

    2002-01-01

    The present study describes the effect of low frequency, low energy, pulsing electromagnetic fields (PEMFs) on A2A adenosine receptors in human neutrophils.Saturation experiments performed using a high affinity adenosine antagonist [3H]-ZM 241385 revealed a single class of binding sites in control and in PEMF-treated human neutrophils with similar affinity (KD=1.05±0.10 and 1.08±0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P<0.01) (Bmax =126±10 and 215±15 fmol mg−1 protein, respectively).The effect of PEMFs was specific to the A2A adenosine receptors. This effect was also intensity, time and temperature dependent.In the adenylyl cyclase assays the A2A receptor agonists, HE-NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC50 value of 43 (40 – 47) and 255 (228 – 284) nM, respectively. The capability of HE-NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P<0.01) after exposure to PEMFs with an EC50 value of 10(8 – 13) and 61(52 – 71) nM, respectively.In the superoxide anion (O2−) production assays HE-NECA and NECA inhibited the generation of O2− in untreated human neutrophils, with an EC50 value of 3.6(3.1 – 4.2) and of 23(20 – 27) nM, respectively. Moreover, in PEMF-treated human neutrophils, the same compounds show an EC50 value of 1.6(1.2 – 2.1) and of 6.0(4.7 – 7.5) nM respectively.These results indicate the presence of significant alterations in the expression and in the functionality of adenosine A2A receptors in human neutrophils treated with PEMFs. PMID:11976268

  4. Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation.

    PubMed

    Seidah, Nabil G; Poirier, Steve; Denis, Maxime; Parker, Rex; Miao, Bowman; Mapelli, Claudio; Prat, Annik; Wassef, Hanny; Davignon, Jean; Hajjar, Katherine A; Mayer, Gaétan

    2012-01-01

    Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a ∼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ∼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by ∼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

  5. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

    PubMed

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K; Blackwell, Timothy S; Xia, Yang; Johnston, Richard A; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R

    2012-06-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A(2B)R) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A(2B)R or treatment with the A(2B)R antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A(2B)R attenuated vascular remodeling and hypertension in our model. Furthermore, direct A(2B)R activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A(2B)R antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.

  6. Physical origins of remarkable thermostabilization by an octuple mutation for the adenosine A2a receptor

    NASA Astrophysics Data System (ADS)

    Kajiwara, Yuta; Ogino, Takahiro; Yasuda, Satoshi; Takamuku, Yuuki; Murata, Takeshi; Kinoshita, Masahiro

    2016-07-01

    It was experimentally showed that the thermal stability of a membrane protein, the adenosine A2a receptor, was remarkably enhanced by an octuple mutation. Here we theoretically prove that the energy decrease arising from the formation of protein intramolecular hydrogen bonds and the solvent-entropy gain upon protein folding are made substantially larger by the mutation, leading to the remarkable enhancement. The solvent is formed by hydrocarbon groups constituting nonpolar chains of the lipid bilayer within a membrane. The mutation modifies geometric characteristics of the structure so that the solvent crowding can be reduced to a larger extent when the protein folds.

  7. FGF acts as a co-transmitter through Adenosine A2A receptor to regulate morphological and physiological synaptic plasticity

    PubMed Central

    Flajolet, Marc; Wang, Zhongfeng; Futter, Marie; Shen, Weixing; Nuangchamnong, Nina; Bendor, Jacob; Palaszewski, Iwona; Nairn, Angus C.; Surmeier, D. James; Greengard, Paul

    2009-01-01

    Summary Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and depression. Adenosine, by activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinson's disease. We report here a direct physical interaction between the G protein-coupled A2A receptor and the receptor tyrosine kinase FGF receptor. Concomitant activation of these two classes of receptors, but not individual activation of either one alone, causes a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity induced by a novel A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions. PMID:18953346

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

    PubMed Central

    Rau, Andrew R.; Ariwodola, Olusegun J.

    2015-01-01

    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 A2A receptor modulation on intrinsic excitability. Results: Activation of adenosine A2A receptors with the selective A2A 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 A2A receptor-mediated effects were blocked by preapplication of a selective A2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A2A 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 A2A 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. PMID:25716780

  9. Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

    PubMed Central

    Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

    2014-01-01

    Background: Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. Methods: C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). Results and conclusions: The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions. PMID:24966910

  10. Behavioural and neurochemical characterization of the adenosine A2A receptor antagonist ST1535.

    PubMed

    Galluzzo, Mariangela; Pintor, Anita; Pèzzola, Antonella; Grieco, Rosa; Borsini, Franco; Popoli, Patrizia

    2008-01-28

    ST1535 (2-butyl-9-methyl-8-(2H-1,2,3-triazol 2-yl)-9 H-purin-6-ylamine) is a novel compound showing a preferential adenosine A(2A) receptor antagonist profile. To explore the potential neuroprotective profile of this compound, we evaluated whether ST1535 prevented quinolinic acid (QA)-induced glutamate outflow in the rat striatum (a reliable index of neuroprotective activity in vivo). Microdialysis experiments were performed in naive Wistar rats. In these experiments, a behaviourally active and inactive doses of ST1535 were used. Both doses significantly prevented QA-induced glutamate outflow in the striatum. These results show that ST1535 protects towards striatal excitotoxicity, even though its reduced A(2A)/A(1) selectivity might limit its actual neuroprotective potential.

  11. Nuclear receptor NR5A2 controls neural stem cell fate decisions during development

    PubMed Central

    Stergiopoulos, Athanasios; Politis, Panagiotis K.

    2016-01-01

    The enormous complexity of mammalian central nervous system (CNS) is generated by highly synchronized actions of diverse factors and signalling molecules in neural stem/progenitor cells (NSCs). However, the molecular mechanisms that integrate extrinsic and intrinsic signals to control proliferation versus differentiation decisions of NSCs are not well-understood. Here we identify nuclear receptor NR5A2 as a central node in these regulatory networks and key player in neural development. Overexpression and loss-of-function experiments in primary NSCs and mouse embryos suggest that NR5A2 synchronizes cell-cycle exit with induction of neurogenesis and inhibition of astrogliogenesis by direct regulatory effects on Ink4/Arf locus, Prox1, a downstream target of proneural genes, as well as Notch1 and JAK/STAT signalling pathways. Upstream of NR5a2, proneural genes, as well as Notch1 and JAK/STAT pathways control NR5a2 endogenous expression. Collectively, these observations render NR5A2 a critical regulator of neural development and target gene for NSC-based treatments of CNS-related diseases. PMID:27447294

  12. Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

    PubMed Central

    Brown, C.M.; Collis, M.G.

    1982-01-01

    1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

  13. Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

    PubMed Central

    Daray, Federico Manuel; Minvielle, Ana Itatí; Puppo, Soledad; Rothlin, Rodolfo Pedro

    2003-01-01

    This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration–response curves to U-46619 (TXA2 mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC50: 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pKB values of 7.96 and 9.07, respectively.Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17-phenyl-trinor-PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17-phenyl-trinor-PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI-192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively.Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed.In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel. PMID:12922927

  14. In vitro metabolism studies of new adenosine A 2A receptor antagonists.

    PubMed

    Marucci, Gabriella; Finaurini, Sara; Buccioni, Michela; Lammi, Carmen; Kandhavelu, Meenakshisundaram; Volpini, Rosaria; Ricciutelli, Massimo; Angeli, Piero; Commandeur, Jan N M; Cristalli, Gloria

    2008-12-01

    Evidence, obtained in rodent and primate models of Parkinson's disease (PD) and in preliminary clinical trials, indicates that adenosine A(2A) receptor antagonists might represent a promising non-dopaminergic therapeutic tool for the treatment of PD. Recently, we have reported the biological evaluation of 8-substituted 9-ethyladenines (ANR) as new A(2A) receptor antagonists, three of which (ANR 82, ANR 94, and ANR 152) showed high efficacy in in vivo models for Parkinson's. Understanding the metabolic pathways of new drug candidates is an important aspect of drug discovery. The ANR compounds have been investigated in order to clarify their activity on rat liver microsomes, and more specifically on recombinant human cytochrome P450 2D6 (CYP2D6). The metabolites of all three compounds were detected by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The results indicate that this class of 9-ethyladenines is metabolized only to a fraction of 1.5-5%. These compounds also act as potent mechanism-based inhibitors of CYP450 and in particular of human isoform CYP2D6. Kinetic-analysis of enzyme inactivation was used to describe the effect of these time-dependent inhibitors and to derive the inhibition parameters K(inact) and K(i) defined with respect to the O-demethylation of dextromethorphan.

  15. Past, present and future of A(2A) adenosine receptor antagonists in the therapy of Parkinson's disease.

    PubMed

    Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E; Franco, Rafael

    2011-12-01

    Several selective antagonists for adenosine A(2A) receptors (A(2A)R) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson's disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D₂ and adenosine A(2A) receptors in the basal ganglia. At present it is believed that A(2A)R antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson's patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A(2A)R antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized antiparkinsonian drug therapy, namely the existence of (hetero)dimers/oligomers of G protein-coupled receptors, a topic that is currently the focus of intense debate within the scientific community. Dopamine D₂ receptors (D₂Rs) expressed in the striatum are known to form heteromers with A(2A) adenosine receptors. Thus, the development of heteromer-specific A(2A) receptor antagonists represents a promising strategy for the identification of more selective and safer drugs.

  16. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  17. Characterization of [125I]ZM 241385 binding to adenosine A2A receptors in the pineal of sheep brain.

    PubMed

    Yan, X; Koos, B J; Kruger, L; Linden, J; Murray, T F

    2006-06-22

    Adenosine is a ubiquitous neuromodulator and homeostatic regulator that exerts its physiologic actions through activation of A(1), A(2A), A(2B) and A(3) adenosine receptor subtypes. In the central nervous system, adenosine's action in neurons is manifested in its modulation of tonic inhibitory control. Adenosine released in the brain during hypoxia has critical depressant effects on breathing in fetal and newborn mammals, an action suggested to be mediated by A(2A) receptors in the posteromedial thalamus. In an effort to more accurately define the spatial distribution of adenosine A(2A) receptors in fetal sheep diencephalon, we have used a receptor autoradiographic technique utilizing an iodinated radioligand [(125)I]ZM 241385, which has greater sensitivity and resolution than the tritiated compound. The distribution of ligand binding sites in the fetal sheep diencephalon indicated that the highest levels of binding were in select thalamic nuclei, including those implicated in hypoxic depression of fetal breathing, and the pineal. Given the high density of labeled A(2A) receptors in the pineal, these sites were characterized more fully in homogenate radioligand binding assays. These data indicate that [(125)I]ZM 241385 binding sites display a pharmacological signature consistent with that of adenosine A(2A) receptors and are expressed at similar levels in fetal, lamb and adult ovine brain. The adenosine A(2A) receptor pharmacologic signature of the [(125)I]ZM 241385 binding site in pineal cell membranes generalized to the site characterized in membranes derived from other portions of the lamb thalamus, including the sector involved in hypoxic inhibition of fetal breathing. These results have important implications for the functional roles of adenosine A(2A) receptors in the thalamus and pineal of sheep brain.

  18. Ligand binding to thromboxane receptors on human platelets: correlation with biological activity.

    PubMed Central

    Armstrong, R. A.; Jones, R. L.; Wilson, N. H.

    1983-01-01

    The preparation of enantiomerically pure [3H]-15 (S) 9, 11-epoxymethano PGH2 (a thromboxane A2-like agonist) has enabled the binding of ligands to the thromboxane receptor of the human platelet to be studied. The binding of the radio-ligand to washed human platelets has 3 components. One component is not displaceable by 'cold' 9, 11-epoxymethano PGH2 and its concentration-binding plot is roughly linear. The other 2 components are displaceable and saturable, and the larger of the two, which is sensitive to the stereochemistry of the C15 secondary alcohol, appears to represent the thromboxane receptor. About 1700 15(S)9, 11-epoxymethano PGH2 molecules are specifically bound to a single platelet and 50% of this binding is achieved with a concentration of 75 nM. Displacement of [3H]-15(S)9, 11-epoxymethano PGH2 is effected by (a) TXA2 and PGH2 and a number of bicyclic stable analogues (e.g. 9,11-azo PGH2), all of which produce irreversible aggregation of human platelets; (b) analogues of PGF2 alpha with potent thromboxane-like activity (e.g. ICI 79939); (c) compounds with partial agonist activity on the platelet thromboxane system (e.g. CTA2); (d) Thromboxane/endoperoxide analogues which specifically antagonize thromboxane-like actions on the human platelet (e.g. PTA2 and EP 045). Displacement is not achieved with the natural prostaglandins PGE2, PGD2 and PGF2 alpha. Neither the thromboxane-synthetase inhibitor dazoxiben nor R(+)-trimethoquinol have high displacing activity. The correlation of radio-ligand displacement with the biological activity of the competing ligands is discussed in relation to the nature of the thromboxane receptor on the human platelet. PMID:6317122

  19. Competitive antagonism at thromboxane receptors in human platelets.

    PubMed Central

    Armstrong, R. A.; Jones, R. L.; Peesapati, V.; Will, S. G.; Wilson, N. H.

    1985-01-01

    The inhibitory effects of three prostanoid analogues, EP 045, EP 092 and pinane thromboxane A2 (PTA2), on the aggregation of human platelets in vitro have been investigated. In diluted platelet-rich plasma (PRP), EP 045 (20 microM) and EP 092 (1 microM) completely inhibited irreversible aggregation responses to thromboxane A2 (TXA2), prostaglandin H2 (PGH2) and five chemically stable thromboxane mimetics, including 11,9-epoxymethano-PGH2 and 9,11-azo-PGH2. Reversible aggregation produced by the prostanoid analogue, CTA2, was also inhibited. The block of the stable agonist action was surmountable. In plasma-free platelet suspensions EP 045 and EP 092 were more potent antagonists. Schild analysis indicated a competitive type of antagonism for EP 045 (affinity constant of 1.1 X 10(7) M-1); the nature of the EP 092 block is not clear. Primary aggregation waves induced by ADP, platelet activating factor (Paf) and adrenaline were unaffected by EP 045 and EP 092, whereas the corresponding second phases of aggregation were suppressed. Aggregation and 5-hydroxytryptamine (5-HT) release induced by either PGH2 or 11,9-epoxymethano-PGH2 were inhibited in a parallel manner by EP 045. Inhibition of thromboxane biosynthesis is not involved in these effects. EP 045 and EP 092 did not raise adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the platelet suspensions. In plasma-free platelet suspensions PTA2 produced a shape change response which could be blocked by EP 045. PTA2, therefore, has a thromboxane-like agonist action. The block of the aggregatory action of 11,9-epoxymethano-PGH2 by PTA2 appears to be mainly due to competition at the thromboxane receptor. However, PTA2 produced a slight rise in cyclic AMP levels; this could be due to a very weak stimulant action on either PGI2 or PGD2 receptors present in the human platelet. Functional antagonism by PTA2 may therefore augment its thromboxane receptor blocking activity. The results are discussed in terms of (a) the

  20. Habit Formation after Random Interval Training Is Associated with Increased Adenosine A2A Receptor and Dopamine D2 Receptor Heterodimers in the Striatum

    PubMed Central

    He, Yan; Li, Yan; Chen, Mozi; Pu, Zhilan; Zhang, Feiyang; Chen, Long; Ruan, Yang; Pan, Xinran; He, Chaoxiang; Chen, Xingjun; Li, Zhihui; Chen, Jiang-Fan

    2016-01-01

    Striatal adenosine A2A receptors (A2ARs) modulate striatal synaptic plasticity and instrumental learning, possibly by functional interaction with the dopamine D2 receptors (D2Rs) and metabotropic glutamate receptors 5 (mGluR5) through receptor-receptor heterodimers, but in vivo evidence for these interactions is lacking. Using in situ proximity ligation assay (PLA), we studied the subregional distribution of the A2AR-D2R and A2AR-mGluR5 heterodimer complexes in the striatum and their adaptive changes over the random interval and random ratio training of instrumental learning. After confirming the specificity of the PLA detection of the A2AR-D2R heterodimers with the A2AR knockout and D2R knockout mice, we detected a heterogeneous distribution of the A2AR-D2R heterodimer complexes in the striatum, being more abundant in the dorsolateral than the dorsomedial striatum. Importantly, habit formation after the random interval training was associated with the increased formation of the A2AR-D2R heterodimer complexes, with prominant increase in the dorsomedial striatum. Conversely, goal-directed behavior after the random ratio schedule was not associated with the adaptive change in the A2AR-D2R heterodimer complexes. In contrast to the A2AR-D2R heterodimers, the A2AR-mGluR5 heterodimers showed neither subregional variation in the striatum nor adaptive changes over either the random ratio (RR) or random interval (RI) training of instrumental learning. These findings suggest that development of habit formation is associated with increased formation of the A2AR-D2R heterodimer protein complexes which may lead to reduced dependence on D2R signaling in the striatum. PMID:28082865

  1. Enhancement of AMPA currents and GluR1 membrane expression through PKA-coupled adenosine A(2A) receptors.

    PubMed

    Dias, Raquel B; Ribeiro, Joaquim A; Sebastião, Ana M

    2012-02-01

    Phosphorylation of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by Protein Kinase A (PKA) is known to regulate AMPA receptor (AMPAR) trafficking and stabilization at the postsynaptic membrane, which in turn is one of the key mechanisms by which synaptic transmission and plasticity are tuned. However, not much is known as to how Gs-coupled receptors contribute to endogenous PKA-mediated regulation of AMPA receptor function. Here we report that activation of the excitatory A(2A) adenosine receptor by 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) facilitates AMPA-evoked currents in CA1 pyramidal neurons, by a mechanism dependent on PKA activation, but not on protein synthesis. This modulation of AMPA currents was mimicked by forskolin (1 μM) and did not occur in stratum radiatum interneurons. Superfusion of the A(2A) receptor agonist also caused an increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs), as well as in the membrane levels of GluR1 subunits phosphorylated at the PKA site (Ser845). The impact of this increase on GluR1-containing AMPA receptor expression was evidenced by the potentiation of LTP at the CA3-CA1 synapse that followed brief activation of A(2A) receptors. We thus propose that in conditions of increased adenosine availability, A(2A) receptor activation is responsible for setting part of the endogenous GluR1 Ser-845 phosphorylation tonus and hence, the availability of the GluR1-containing AMPA receptor extrasynaptic pool for synaptic insertion and reinforcement of synaptic strength.

  2. Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects

    PubMed Central

    2011-01-01

    Background Permanent functional deficits following spinal cord injury (SCI) arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites. Methods Spinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord. Results SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI), reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK) 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours), these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the neuroprotective effects

  3. New adenosine A2A receptor antagonists: actions on Parkinson's disease models.

    PubMed

    Pinna, Annalisa; Volpini, Rosaria; Cristalli, Gloria; Morelli, Micaela

    2005-04-11

    The 8-substituted 9-ethyladenine derivatives: 8-bromo-9-ethyladenine (ANR 82), 8-ethoxy- 9-ethyladenine (ANR 94), and 8-furyl-9-ethyladenine (ANR 152) have been characterized in vitro as adenosine receptor antagonists. Adenosine is deeply involved in the control of motor behaviour and substantial evidences indicate that adenosine A(2A) receptor antagonists improve motor deficits in animal models of Parkinson's disease. On this basis, the efficacy of ANR 82, ANR 94, and ANR 152 in rat models of Parkinson's disease was evaluated. All compounds tested reversed the catalepsy induced by haloperidol. However, in unilaterally 6-hydroxydopamine-lesioned rats, only ANR 94 and ANR 152 potentiated l-dihydroxy-phenylalanine (l-DOPA) effect on turning behaviour and induced contralateral turning behaviour in rats sensitised to l-DOPA. Taken together the results of this study indicate that some 8-substituted 9-ethyladenine derivatives ameliorate motor deficits in rat models of Parkinson's disease, suggesting a potential therapeutic role of these compounds.

  4. A tail of two signals: the C terminus of the A(2A)-adenosine receptor recruits alternative signaling pathways.

    PubMed

    Gsandtner, Ingrid; Freissmuth, Michael

    2006-08-01

    G protein-coupled receptors are endowed with carboxyl termini that vary greatly in length and sequence. In most instances, the distal portion of the C terminus is dispensable for G protein coupling. This is also true for the A(2A)-adenosine receptor, where the last 100 amino acids are of very modest relevance to G(s) coupling. The C terminus was originally viewed mainly as the docking site for regulatory proteins of the beta-arrestin family. These beta-arrestins bind to residues that have been phosphorylated by specialized kinases (G protein-coupled receptor kinases) and thereby initiate receptor desensitization and endocytosis. More recently, it has become clear that many additional "accessory" proteins bind to C termini of G protein-coupled receptors. The article by Sun et al. in the current issue of Molecular Pharmacology identifies translin-associated protein-X as yet another interaction partner of the A(2A) receptor; translin-associated protein allows the A(2A) receptor to impinge on the signaling mechanisms by which p53 regulates neuronal differentiation, but the underlying signaling pathways are uncharted territory. With a list of five known interaction partners, the C terminus of the A(2A) receptor becomes a crowded place. Hence, there must be rules that regulate the interaction. This allows the C terminus to act as coincidence detector and as signal integrator. Despite our ignorance about the precise mechanisms, the article has exciting implications: the gene encoding for translin-associated protein-X maps to a locus implicated in some forms of schizophrenia; A(2A) receptor agonists are candidate drugs for the treatment of schizophrenic symptoms. It is of obvious interest to explore a possible link.

  5. Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism.

    PubMed

    Zanos, Panos; Wright, Sherie R; Georgiou, Polymnia; Yoo, Ji Hoon; Ledent, Catherine; Hourani, Susanna M; Kitchen, Ian; Winsky-Sommerer, Raphaelle; Bailey, Alexis

    2014-04-01

    There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4ml/kg/day, i.p.) or methamphetamine (1mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [(125)I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A2A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A2A receptors which were chronically treated with methamphetamine (1mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor

  6. Phospholipase A2 receptor positive membranous nephropathy long after living donor kidney transplantation between identical twins.

    PubMed

    Saito, Hisako; Hamasaki, Yoshifumi; Tojo, Akihiro; Shintani, Yukako; Shimizu, Akira; Nangaku, Masaomi

    2015-07-01

    Although membranous nephropathy (MN) is a commonly observed cause of post-transplant glomerulonephritis, distinguishing de novo from recurrent MN in kidney allograft is often difficult. Phospholipase A2 receptor (PLA2R) staining is useful for diagnosing recurrent MN in allografts similarly to idiopathic MN in native kidney. No specific treatment strategy has been established for MN, especially when accompanied with HCV infection in kidney transplant recipients. This report describes a 66-year-old man who was diagnosed as having PLA2R positive membranous nephropathy accompanied with already-known IgA nephropathy and HCV infection 26 years after kidney transplantation conducted between identical twins. PLA2R was detected along capillary loops, implying that this patient is affected by the same pathogenic mechanism as idiopathic MN, not secondary MN associated with other disorders such as HCV infection. The patient successfully achieved clinical remission after steroid therapy.

  7. Antiphospholipase A2 Receptor Autoantibodies: A Step Forward in the Management of Primary Membranous Nephropathy

    PubMed Central

    Obrisca, Bogdan; Ismail, Gener; Jurubita, Roxana; Baston, Catalin; Andronesi, Andreea; Mircescu, Gabriel

    2015-01-01

    Since the identification of PLA2R (M-type phospholipase A2 receptor) as the first human antigenic target in primary membranous nephropathy (MN), perpetual progress has been made in understanding the pathogenesis of this disease. Accumulating clinical data support a pathogenic role for the anti-PLA2R antibodies (PLA2R ABs), but confirmation in an animal model is still lacking. However, PLA2R ABs were related to disease activity and outcome, as well as to response therapy. Accordingly, PLA2R ABs assay seems to be promising tool not only to diagnose MN but also to predict the course of the disease and could open the way to personalize therapy. Nevertheless, validation of a universal assay with high precision and definition of cut-off levels, followed by larger studies with a prolonged follow-up period, are needed to confirm these prospects. PMID:26576418

  8. Adenosine A(2A)-cannabinoid CB(1) receptor interaction: an integrative mechanism in striatal glutamatergic neurotransmission.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Popoli, Patrizia

    2012-10-02

    The striatum is a subcortical area involved in sensorimotor, cognitive and emotional processes. Adenosine A(2A) receptors (A(2A)Rs) are highly expressed in the striatum, and their ability to establish functional and molecular interactions with many other receptors attributes to a pivotal role in the modulation and integration of striatal neurotransmission. This review will focus on the interaction between A(2A)Rs and cannabinoid CB(1) receptors (CB(1)Rs), taking it as a paradigmatic example of synaptic integration. Indeed, A(2A)Rs can exert an opposite (permissive vs. inhibitory) influence on CB1-dependent synaptic effect. These apparently irreconcilable functions could depend on a different role of pre- vs. postsynaptic A(2A)Rs, on their interaction with other receptors (namely adenosine A(1), metabotropic glutamate 5 and dopamine D2 receptors), and on whether A(2A)Rs form or not heteromers with CB(1)Rs. Besides providing a good example of the intricate pattern of events taking place in striatal synapses, the A(2A)/CB(1)R interaction proves very informative to understand the physiology of the basal ganglia and the mechanisms of related diseases. This article is part of a Special Issue entitled: Brain Integration.

  9. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease. PMID:28167788

  10. Architecture of fully occupied GluA2 AMPA receptor-TARP complex elucidated by cryo-EM.

    PubMed

    Zhao, Yan; Chen, Shanshuang; Yoshioka, Craig; Baconguis, Isabelle; Gouaux, Eric

    2016-08-04

    Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates receptor trafficking and the kinetics of receptor gating and pharmacology. Here we report the cryo-electron microscopy (cryo-EM) structure of the homomeric rat GluA2 AMPA receptor saturated with TARP γ2 subunits, which shows how the TARPs are arranged with four-fold symmetry around the ion channel domain and make extensive interactions with the M1, M2 and M4 transmembrane helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand-binding domain (LBD) 'clamshells', one pair of TARPs is juxtaposed near the LBD dimer interface, whereas the other pair is near the LBD dimer-dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD clamshell closure, but also affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane domains buttress the ion channel pore.

  11. Alteration in contractile G-protein coupled receptor expression by moist snus and nicotine in rat cerebral arteries

    SciTech Connect

    Sandhu, Hardip; Xu Cangbao; Edvinsson, Lars

    2011-04-15

    The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET{sub B}, serotonin 5-HT{sub 1B}, and thromboxane A{sub 2} TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24 h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25 ng nicotine/ml). A high dose (250 ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c, 5-HT{sub 1B} receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET{sub B}, 5-HT{sub 1B}, and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25 ng nicotine/ml) lowered the 5-HT{sub 1B} receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250 ng nicotine/ml) increased the ET{sub B} receptor-mediated contraction. The combined 5-HT{sub 1B} and 5-HT{sub 2A} receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or

  12. A2A adenosine receptor regulates the human blood brain barrier permeability

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2015-01-01

    The blood brain barrier (BBB) symbolically represents the gateway to the central nervous system. It is a single layer of specialized endothelial cells that coats the central nervous system (CNS) vasculature and physically separates the brain environment from the blood constituents, to maintain the homeostasis of the CNS. However, this protective measure is a hindrance to the delivery of therapeutics to treat neurological diseases. Here, we show that activation of A2A adenosine receptor (AR) with an FDA-approved agonist potently permeabilizes an in vitro primary human brain endothelial barrier (hBBB) to the passage of chemotherapeutic drugs and T cells. T cell migration under AR signaling occurs primarily by paracellular transendothelial route. Permeabilization of the hBBB is rapid, time-dependent and reversible and is mediated by morphological changes in actin-cytoskeletal reorganization induced by RhoA signaling and a potent down-regulation of Claudin-5 and VE-Cadherin. Moreover, the kinetics of BBB permeability in mice closely overlaps with the permeability kinetics of the hBBB. These data suggest that activation of A2A AR is an endogenous mechanism that may be used for CNS drug delivery in human. PMID:25262373

  13. Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre- and postsynaptic sites in the rat striatum.

    PubMed

    Tebano, Maria Teresa; Pintor, Annita; Frank, Claudio; Domenici, Maria Rosaria; Martire, Alberto; Pepponi, Rita; Potenza, Rosa Luisa; Grieco, Rosa; Popoli, Patrizia

    2004-07-01

    Adenosine A(2A) receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A(2A) receptor antagonists in models of pre- and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A(2A) receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose-dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30-100 nM) significantly reduced 4-aminopyridine (4-AP)-induced paired-pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N-methyl-D-aspartate (NMDA; 12.5 and 50 microM). The A(2A) antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A(2A) agonist CGS 21680 (100 nM) showed a protective influence toward 50 microM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca(2+)](i) or the release of lactate dehydrogenase (LDH) induced by 100 and 300 microM NMDA, respectively. The ability of ZM 241385 to prevent QA-induced glutamate outflow and 4-AP-induced effects confirms that A(2A) receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA-induced effects suggest that A(2A) receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A(2A) antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.

  14. A2A receptor antagonists do not induce dyskinesias in drug-naive or L-dopa sensitized rats.

    PubMed

    Jones, N; Bleickardt, C; Mullins, D; Parker, E; Hodgson, R

    2013-09-01

    L-dopa, the precursor to dopamine, is currently the gold standard treatment for Parkinson's disease (PD). However, chronic exposure is associated with L-dopa-induced dyskinesias (LIDs), a serious side effect characterized by involuntary movements. Adenosine A2A receptor antagonists have been studied as a novel non-dopaminergic PD treatment. Because A2A receptor antagonists do not act on dopamine receptors, it has been hypothesized that they will not induce dyskinesias characteristic of L-dopa. To test this hypothesis in a rodent model, the A2A receptor antagonists SCH 412348 (3 mg/kg), vipadenant (10 mg/kg), caffeine (30 mg/kg), or istradefylline (3 mg/kg) were chronically (19-22 days) administered to Sprague Dawley rats, and dyskinetic behaviors were scored across this chronic dosing paradigm. Unlike L-dopa, there was no evidence of dyskinetic activity resulting from any of the four A2A receptor antagonists tested. When delivered to animals previously sensitized with L-dopa (6 mg/kg), SCH 412348, vipadenant, caffeine or istradefylline treatment produced no dyskinesias. When administered in combination with L-dopa (6 mg/kg), SCH 412348 (3 mg/kg) neither exacerbated nor prevented the induction of LIDs over the course of 19 days of treatment. Collectively, our data indicate that A2A receptor antagonists are likely to have a reduced dyskinetic liability relative to L-dopa but do not block dyskinesias when coadministered with L-dopa. Clinical studies are required to fully understand the dyskinesia profiles of A2A receptor antagonists.

  15. Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.

    PubMed

    Doré, Andrew S; Robertson, Nathan; Errey, James C; Ng, Irene; Hollenstein, Kaspar; Tehan, Ben; Hurrell, Edward; Bennett, Kirstie; Congreve, Miles; Magnani, Francesca; Tate, Christopher G; Weir, Malcolm; Marshall, Fiona H

    2011-09-07

    Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

  16. Discovery and optimization of potent and selective functional antagonists of the human adenosine A2B receptor.

    PubMed

    Bedford, Simon T; Benwell, Karen R; Brooks, Teresa; Chen, Ijen; Comer, Mike; Dugdale, Sarah; Haymes, Tim; Jordan, Allan M; Kennett, Guy A; Knight, Anthony R; Klenke, Burkhard; LeStrat, Loic; Merrett, Angela; Misra, Anil; Lightowler, Sean; Padfield, Anthony; Poullennec, Karine; Reece, Mark; Simmonite, Heather; Wong, Melanie; Yule, Ian A

    2009-10-15

    We herein report the discovery of a novel class of antagonists of the human adenosine A2B receptor. This low molecular weight scaffold has been optimized to offer derivatives with potential utility for the alleviation of conditions associated with this receptor subtype, such as nociception, diabetes, asthma and COPD. Furthermore, preliminary pharmacokinetic analysis has revealed compounds with profiles suitable for either inhaled or systemic routes of administration.

  17. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    PubMed Central

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease. PMID:26796668

  18. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  19. Influence of CGS 21680, a selective adenosine A(2A) receptor agonist, on NMDA receptor function and expression in the brain of Huntington's disease mice.

    PubMed

    Ferrante, Antonella; Martire, Alberto; Armida, Monica; Chiodi, Valentina; Pézzola, Antonella; Potenza, Rosa Luisa; Domenici, Maria Rosaria; Popoli, Patrizia

    2010-04-06

    The effect of chronic treatment with the selective adenosine A(2A) receptor agonist CGS 21680 on N-Methyl-d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntington's disease (HD). Starting from 8weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5mg/kg i.p.) for 3weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A(2A) receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.

  20. Neuroprotection by caffeine in the MPTP model of parkinson's disease and its dependence on adenosine A2A receptors.

    PubMed

    Xu, K; Di Luca, D G; Orrú, M; Xu, Y; Chen, J-F; Schwarzschild, M A

    2016-05-13

    Considerable epidemiological and laboratory data have suggested that caffeine, a nonselective adenosine receptor antagonist, may protect against the underlying neurodegeneration of parkinson's disease (PD). Although both caffeine and more specific antagonists of the A2A subtype of adenosine receptor (A2AR) have been found to confer protection in animal models of PD, the dependence of caffeine's neuroprotective effects on the A2AR is not known. To definitively determine its A2AR dependence, the effect of caffeine on 1-methyl-4-phenyl-1,2,3,6 tetra-hydropyridine (MPTP) neurotoxicity was compared in wild-type (WT) and A2AR gene global knockout (A2A KO) mice, as well as in central nervous system (CNS) cell type-specific (conditional) A2AR knockout (cKO) mice that lack the receptor either in postnatal forebrain neurons or in astrocytes. In WT and in heterozygous A2AR KO mice caffeine pretreatment (25mg/kgip) significantly attenuated MPTP-induced depletion of striatal dopamine. By contrast in homozygous A2AR global KO mice caffeine had no effect on MPTP toxicity. In forebrain neuron A2AR cKO mice, caffeine lost its locomotor stimulant effect, whereas its neuroprotective effect was mostly preserved. In astrocytic A2AR cKO mice, both caffeine's locomotor stimulant and protective properties were undiminished. Taken together, these results indicate that neuroprotection by caffeine in the MPTP model of PD relies on the A2AR, although the specific cellular localization of these receptors remains to be determined.

  1. Caffeine and adenosine A(2a) receptor antagonists prevent beta-amyloid (25-35)-induced cognitive deficits in mice.

    PubMed

    Dall'Igna, Oscar P; Fett, Paulo; Gomes, Marcio W; Souza, Diogo O; Cunha, Rodrigo A; Lara, Diogo R

    2007-01-01

    Consumption of caffeine, an adenosine receptor antagonist, was found to be inversely associated with the incidence of Alzheimer's disease. Moreover, caffeine protects cultured neurons against beta-amyloid-induced toxicity, an effect mimicked by adenosine A(2A) but not A(1) receptor antagonists. We now tested if caffeine administration would prevent beta-amyloid-induced cognitive impairment in mice and if this was mimicked by A(2A) receptor blockade. One week after icv administration of the 25-35 fragment of beta-amyloid (Abeta, 3 nmol), mice displayed impaired performance in both inhibitory avoidance and spontaneous alternation tests. Prolonged treatment with caffeine (1 mg/ml) had no effect alone but prevented the Abeta-induced cognitive impairment in both tasks when associated with acute caffeine (30 mg/kg) 30 min treatment before Abeta administration. The same protective effect was observed after subchronic (4 days) treatment with daily injections of either caffeine (30 mg/kg) or the selective adenosine A(2A) receptor antagonist SCH58261 (0.5 mg/kg). This provides the first direct in vivo evidence that caffeine and A(2A) receptor antagonists afford a protection against Abeta-induced amnesia, which prompts their interest for managing Alzheimer's disease.

  2. Enigma (partially) resolved: phospholipase A2 receptor is the cause of "idiopathic" membranous glomerulonephritis.

    PubMed

    Truong, Luan D; Seshan, Surya V

    2015-12-15

    Membranous glomerulonephritis (MGN) is a very significant kidney disease. It is one of the frequent causes of heavy protein excretion in urine. MGN is thought to be an immune-mediated disease caused by glomerular deposition of antigen-antibody complexes. The pathogenic antigen, however, has been an enigma until recently. It was discovered in 2009 that phospholipase A2 receptor (PLA2R), a normal transmembrane protein in podocyte plasma membrane, is the antigen causing MGN. Within 5 yr of its discovery, this seminal finding has leaded to novel insights into the treatment of this disease including diagnosis, therapy, and prediction of outcome. This finding also paves the way for fundamental studies on how and why autoimmunity against PLA2R develops. The discovery of PLA2A as the cause of "idiopathic" MGN after a half century of speculation, followed by further fundamental insights with such an expedient and successful application in patient care, embodies the elegance of science at its junction with society. This perspective traces the story of this remarkable discovery.

  3. A differential role for the adenosine A2A receptor in opiate reinforcement vs opiate-seeking behavior.

    PubMed

    Brown, Robyn Mary; Short, Jennifer Lynn; Cowen, Michael Scott; Ledent, Catherine; Lawrence, Andrew John

    2009-03-01

    The adenosine A(2A) receptor is specifically enriched in the medium spiny neurons that make up the 'indirect' output pathway from the ventral striatum, a structure known to have a crucial, integrative role in processes such as reward, motivation, and drug-seeking behavior. In the present study we investigated the impact of adenosine A(2A) receptor deletion on behavioral responses to morphine in a number of reward-related paradigms. The acute, rewarding effects of morphine were evaluated using the conditioned place preference paradigm. Operant self-administration of morphine on both fixed and progressive ratio schedules as well as cue-induced drug-seeking was assessed. In addition, the acute locomotor response to morphine as well as sensitization to morphine was evaluated. Decreased morphine self-administration and breakpoint in A(2A) knockout mice was observed. These data support a decrease in motivation to consume the drug, perhaps reflecting diminished rewarding effects of morphine in A(2A) knockout mice. In support of this finding, a place preference to morphine was not observed in A(2A) knockout mice but was present in wild-type mice. In contrast, robust cue-induced morphine-seeking behavior was exhibited by both A(2A) knockout and wild-type mice after a period of withdrawal. The acute locomotor response to morphine in the A(2A) knockout was similar to wild-type mice, yet A(2A) knockout mice did not display tolerance to chronic morphine under the present paradigm. Both genotypes display locomotor sensitization to morphine, implying a lack of a role for the A(2A) receptor in the drug-induced plasticity necessary for the development or expression of sensitization. Collectively, these data suggest a differential role for adenosine A(2A) receptors in opiate reinforcement compared to opiate-seeking.

  4. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  5. Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

    PubMed

    Segala, Elena; Guo, Dong; Cheng, Robert K Y; Bortolato, Andrea; Deflorian, Francesca; Doré, Andrew S; Errey, James C; Heitman, Laura H; IJzerman, Adriaan P; Marshall, Fiona H; Cooke, Robert M

    2016-07-14

    The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

  6. Increased non-rapid eye movement sleep by cocaine withdrawal: possible involvement of A2A receptors.

    PubMed

    Yang, Shu-Long; Han, Jin-Yi; Kim, Yun-Bae; Nam, Sang-Yoon; Song, Sukgil; Hong, Jin Tae; Oh, Ki-Wan

    2011-02-01

    This study attempted to clarify whether cocaine withdrawal altered sleep architecture and the role of adenosine receptors in this process. Cocaine (20 mg/kg) was administered subcutaneously once per day for 7 days to rat implanted with sleep/wake recording electrode. Polygraphic signs of undisturbed sleep/wake activities were recorded for 24 h before cocaine administration (basal recording as control); withdrawal-day 1 (after 1 day of repeated cocaine administration), withdrawal-day 8 (after 8 days of repeated cocaine administration), and withdrawal-day 14 (after 14 days of repeated cocaine administration), respectively. On cocaine withdrawal-day 1, wakefulness was significantly increased, total sleep was decreased, non-rapid eye movement sleep was markedly reduced, and rapid eye movement sleep was enhanced. Sleep/wake cycles were also increased on cocaine withdrawal day 1. However, non-rapid eye movement sleep was increased on withdrawal-day 8 and 14, whereas rapid eye movement sleep was decreased and no significant changes were observed in the total sleep and sleep/wake cycles during these periods. Adenosine A(2A) receptors expression was increased on withdrawal-day 8 and 14, whereas A(1) receptors levels were reduced after 14 days of withdrawal and the A(2B) receptors remained unchanged. Our findings suggest that alterations of sleep and sleep architecture during cocaine subacute and subchronic withdrawals after repeated cocaine administration may be partially involved in A(2A) receptors over-expression in the rat hypothalamus.

  7. The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6.

    PubMed

    Wilson, Jeffrey M; Kurtz, Courtney C; Black, Steven G; Ross, William G; Alam, Mohammed S; Linden, Joel; Ernst, Peter B

    2011-06-15

    Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4(+) T cell regulation, we report that adenosine acts via dendritic cell (DC) A(2B) adenosine receptor (A(2B)AR) to promote the development of Th17 cells. Mouse naive CD4(+) T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5'-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17- and IL-22-secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A(2B)AR(-/-) or A(2A)AR(-/-)/A(2B)AR(-/-) mice indicated that the DC A(2B)AR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4(+) T cell differentiation and suggested a mechanism for putative proinflammatory effects of A(2B)AR.

  8. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGES

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; ...

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, ormore » DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  9. Brain-Penetrant Tetrahydronaphthalene Thromboxane A2-Prostanoid (TP) Receptor Antagonists as Prototype Therapeutics for Alzheimer’s Disease

    PubMed Central

    2012-01-01

    A hallmark pathological feature of the Alzheimer’s disease (AD) brain is the presence of senile plaques, which comprise amyloid β (Aβ) peptides that are derived from the amyloid precursor protein (APP). The plaque-containing AD brain is thought to be under oxidative stress, as evidenced by increased lipid oxidation products that include isoprostane-F2αIII (iPF2αIII). IPF2αIII can bind to and activate the thromboxane A2-prostanoid (TP) receptor, and TP receptor activation causes increased Aβ production through enhancement of APP mRNA stability. Moreover, TP receptor antagonists have been shown to block iPF2αIII-induced increases of Aβ secretion. Thus, the TP receptor may be a potential drug target for AD therapy. However, here we show that existing TP receptor antagonists have poor blood-brain barrier (BBB) permeability, likely due to the presence of a carboxylic acid moiety that is believed to be important for receptor interaction, but which may hamper passive diffusion across the BBB. We now report selected analogues of a known tetrahydronaphthalene TP receptor antagonist, wherein the carboxylic acid moiety has been replaced by heterocyclic bioisosteres. These heterocyclic analogues retained relatively high affinity for the mouse and human TP receptors, and, unlike the parent carboxylic acid compound, several examples freely diffused across the BBB into the brain upon administration to mice. These results reveal that brain-penetrant tetrahydronaphthalene TP receptor antagonists can be developed by substituting the carboxylic acid moiety with a suitable nonacidic bioisostere. Compounds of this type hold promise as potential lead structures to develop drug candidates for the treatment of AD. PMID:23173073

  10. Crystal structure of the BoNT/A2 receptor-binding domain in complex with the luminal domain of its neuronal receptor SV2C

    PubMed Central

    Benoit, Roger M.; Schärer, Martin A.; Wieser, Mara M.; Li, Xiaodan; Frey, Daniel; Kammerer, Richard A.

    2017-01-01

    A detailed molecular understanding of botulinum neurotoxin (BoNT)/host-cell-receptor interactions is fundamental both for developing strategies against botulism and for generating improved BoNT variants for medical applications. The X-ray crystal structure of the receptor-binding domain (HC) of BoNT/A1 in complex with the luminal domain (LD) of its neuronal receptor SV2C revealed only few specific side-chain – side-chain interactions that are important for binding. Notably, two BoNT/A1 residues, Arg 1156 and Arg 1294, that are crucial for the interaction with SV2, are not conserved among subtypes. Because it has been suggested that differential receptor binding of subtypes might explain their differences in biological activity, we determined the crystal structure of BoNT/A2-HC in complex with SV2C-LD. Although only few side-chain interactions are conserved between the two BoNT/A subtypes, the overall binding mode of subtypes A1 and A2 is virtually identical. In the BoNT/A2-HC – SV2C complex structure, a missing cation-π stacking is compensated for by an additional salt bridge and an anion-π stacking interaction, which explains why the binding of BoNT/A subtypes to SV2C tolerates variable side chains. These findings suggest that motif extensions and a shallow binding cleft in BoNT/A-HC contribute to binding specificity. PMID:28252640

  11. M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy

    PubMed Central

    Beck, Laurence H.; Bonegio, Ramon G.B.; Lambeau, Gérard; Beck, David M.; Powell, David W.; Cummins, Timothy D.; Klein, Jon B.; Salant, David J.

    2009-01-01

    BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in non-reduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A2 receptor (PLA2R). Reactive serum specimens recognized recombinant PLA2R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA2R antibody. Anti-PLA2R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA2R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA2R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA2R. PLA2R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA2R is a major antigen in this disease. PMID:19571279

  12. Blunted dynamics of adenosine A2A receptors is associated with increased susceptibility to Candida albicans infection in the elderly

    PubMed Central

    Rodrigues, Lisa; Miranda, Isabel M.; Andrade, Geanne M.; Mota, Marta; Cortes, Luísa; Rodrigues, Acácio G.; Cunha, Rodrigo A.; Gonçalves, Teresa

    2016-01-01

    Opportunistic gut infections and chronic inflammation, in particular due to overgrowth of Candida albicans present in the gut microbiota, are increasingly reported in the elder population. In aged, adult and young mice, we now compared the relative intestinal over-colonization by ingested C. albicans and their translocation to other organs, focusing on the role of adenosine A2A receptors that are a main stop signal of inflammation. We report that elderly mice are more prone to over-colonization by C. albicans than adult and young mice. This fungal over-growth seems to be related with higher growth rate in intestinal lumen, independent of gut tissues invasion, but resulting in higher GI tract inflammation. We observed a particularly high colonization of the stomach, with increased rate of yeast-to-hypha transition in aged mice. We found a correlation between A2A receptor density and tissue damage due to yeast infection: comparing with young and adults, aged mice have a lower gut A2A receptor density and C. albicans infection failed to increase it. In conclusion, this study shows that aged mice have a lower ability to cope with inflammation due to C. albicans over-colonization, associated with an inability to adaptively adjust adenosine A2A receptors density. PMID:27590517

  13. Solution structure of the first Sam domain of Odin and binding studies with the EphA2 receptor.

    PubMed

    Mercurio, Flavia Anna; Marasco, Daniela; Pirone, Luciano; Pedone, Emilia Maria; Pellecchia, Maurizio; Leone, Marilisa

    2012-03-13

    The EphA2 receptor plays key roles in many physiological and pathological events, including cancer. The process of receptor endocytosis and the consequent degradation have attracted attention as possible means of overcoming the negative outcomes of EphA2 in cancer cells and decreasing tumor malignancy. A recent study indicates that Sam (sterile alpha motif) domains of Odin, a member of the ANKS (ankyrin repeat and sterile alpha motif domain-containing) family of proteins, are important for the regulation of EphA2 endocytosis. Odin contains two tandem Sam domains (Odin-Sam1 and -Sam2). Herein, we report on the nuclear magnetic resonance (NMR) solution structure of Odin-Sam1; through a variety of assays (employing NMR, surface plasmon resonance, and isothermal titration calorimetry techniques), we clearly demonstrate that Odin-Sam1 binds to the Sam domain of EphA2 in the low micromolar range. NMR chemical shift perturbation experiments and molecular modeling studies point out that the two Sam domains interact with a head-to-tail topology characteristic of several Sam-Sam complexes. This binding mode is similar to that we have previously proposed for the association between the Sam domains of the lipid phosphatase Ship2 and EphA2. This work further validates structural elements relevant for the heterotypic Sam-Sam interactions of EphA2 and provides novel insights for the design of potential therapeutic compounds that can modulate receptor endocytosis.

  14. A new ethyladenine antagonist of adenosine A(2A) receptors: behavioral and biochemical characterization as an antiparkinsonian drug.

    PubMed

    Pinna, Annalisa; Tronci, Elisabetta; Schintu, Nicoletta; Simola, Nicola; Volpini, Rosaria; Pontis, Silvia; Cristalli, Gloria; Morelli, Micaela

    2010-03-01

    Adenosine A(2A) receptor antagonists have emerged as an attractive non-dopaminergic target in clinical trials aimed at evaluating improvement in motor deficits in Parkinson's disease (PD). Moreover, preclinical studies suggest that A(2A) receptor antagonists may slow the course of the underlying neurodegeneration of dopaminergic neurons. In this study, we evaluated the efficacy of the new adenosine A(2A) receptor antagonist 8-ethoxy-9-ethyladenine (ANR 94) in parkinsonian models of akinesia and tremor. In addition, induction of the immediate early gene zif-268, and neuroprotective and anti-inflammatory effects of ANR 94 were evaluated. ANR 94 was effective in reversing parkinsonian tremor induced by the administration of tacrine. ANR 94 also counteracted akinesia (stepping test) and sensorimotor deficits (vibrissae-elicited forelimb-placing test), as well as potentiating l-dopa-induced contralateral turning behavior in 6-hydroxydopamine (6-OHDA) lesion model of PD. Potentiation of motor behavior in 6-OHDA-lesioned rats was not associated with increased induction of the immediate early gene zif-268 in the striatum, suggesting that ANR 94 does not induce long-term plastic changes in this structure. Finally, in a subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, ANR 94 protected nigrostriatal dopaminergic neurons from degeneration and counteracted neuroinflammatory processes by contrasting astroglial (glial fibrillary acidic protein, GFAP) and microglial (CD11b) activation. A(2A) receptor antagonism represents a uniquely realistic opportunity for improving PD treatment, since A(2A) receptor antagonists offer substantial symptomatic benefits and possibly disease-modifying activity. The characterization of ANR 94 may represent a further therapeutic opportunity for the treatment of PD with this new class of drugs.

  15. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury

    PubMed Central

    Gazoni, Leo M.; Walters, Dustin M.; Unger, Eric B.; Linden, Joel; Kron, Irving L.; Laubach, Victor E.

    2010-01-01

    Objective Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion (IR) injury. We hypothesized that activation of A1, A2A, or A3 adenosine receptors would provide protection against lung IR injury. Methods Using an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours cold ischemia followed by 2 hours reperfusion. Lungs were administered either vehicle, adenosine, or selective A1, A2A, or A3 receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion. Results Compared to the vehicle-treated control group, treatment with A1, A2A, or A3 agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced TNF-α production. Adenosine treatment was also protective but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A2A agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A1 or A3 agonists. Conclusions Selective activation of A1, A2A, or A3 adenosine receptors provides significant protection against lung IR injury. The decreased elaboration of the potent proinflammatory cytokine, TNF-α, and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients. PMID:20398911

  16. A2A Adenosine Receptor Antagonism Reverts the Blood-Brain Barrier Dysfunction Induced by Sleep Restriction

    PubMed Central

    Hurtado-Alvarado, Gabriela; Domínguez-Salazar, Emilio; Velázquez-Moctezuma, Javier

    2016-01-01

    Chronic sleep restriction induces blood-brain barrier disruption and increases pro-inflammatory mediators in rodents. Those inflammatory mediators may modulate the blood-brain barrier and constitute a link between sleep loss and blood-brain barrier physiology. We propose that adenosine action on its A2A receptor may be modulating the blood-brain barrier dynamics in sleep-restricted rats. We administrated a selective A2A adenosine receptor antagonist (SCH58261) in sleep-restricted rats at the 10th day of sleep restriction and evaluated the blood-brain barrier permeability to dextrans coupled to fluorescein (FITC-dextrans) and Evans blue. In addition, we evaluated by western blot the expression of tight junction proteins (claudin-5, occludin, ZO-1), adherens junction protein (E-cadherin), A2A adenosine receptor, adenosine-synthesizing enzyme (CD73), and neuroinflammatory markers (Iba-1 and GFAP) in the cerebral cortex, hippocampus, basal nuclei and cerebellar vermis. Sleep restriction increased blood-brain barrier permeability to FITC-dextrans and Evans blue, and the effect was reverted by the administration of SCH58261 in almost all brain regions, excluding the cerebellum. Sleep restriction increased the expression of A2A adenosine receptor only in the hippocampus and basal nuclei without changing the expression of CD73 in all brain regions. Sleep restriction reduced the expression of tight junction proteins in all brain regions, except in the cerebellum; and SCH58261 restored the levels of tight junction proteins in the cortex, hippocampus and basal nuclei. Finally, sleep restriction induced GFAP and Iba-1 overexpression that was attenuated with the administration of SCH58261. These data suggest that the action of adenosine on its A2A receptor may have a crucial role in blood-brain barrier dysfunction during sleep loss probably by direct modulation of brain endothelial cell permeability or through a mechanism that involves gliosis with subsequent inflammation and

  17. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  18. Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

    PubMed

    Sablin, Elena P; Blind, Raymond D; Uthayaruban, Rubatharshini; Chiu, Hsiu-Ju; Deacon, Ashley M; Das, Debanu; Ingraham, Holly A; Fletterick, Robert J

    2015-12-01

    The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

  19. Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation.

    PubMed

    Mouro, Francisco M; Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Baqi, Younis; Müller, Christa E; Lopes, Luísa V; Ribeiro, Joaquim A; Sebastião, Ana M

    2017-05-01

    Cannabinoid-mediated memory impairment is a concern in cannabinoid-based therapies. Caffeine exacerbates cannabinoid CB1 receptor (CB1R)-induced memory deficits through an adenosine A1 receptor-mediated mechanism. We now evaluated how chronic or acute blockade of adenosine A2A receptors (A2ARs) affects long-term episodic memory deficits induced by a single injection of a selective CB1R agonist. Long-term episodic memory was assessed by the novel object recognition (NOR) test. Mice received an intraperitoneal (i.p.) injection of the CB1/CB2 receptor agonist WIN 55,212-2 (1 mg/kg) immediately after the NOR training, being tested for novelty recognition 24 h later. Anxiety levels were assessed by the Elevated Plus Maze test, immediately after the NOR. Mice were also tested for exploratory behaviour at the Open Field. For chronic A2AR blockade, KW-6002 (istradefylline) (3 mg/kg/day) was administered orally for 30 days; acute blockade of A2ARs was assessed by i.p. injection of SCH 58261 (1 mg/kg) administered either together with WIN 55,212-2 or only 30 min before the NOR test phase. The involvement of CB1Rs was assessed by using the CB1R antagonist, AM251 (3 mg/kg, i.p.). WIN 55,212-2 caused a disruption in NOR, an action absent in mice also receiving AM251, KW-6002 or SCH 58261 during the encoding/consolidation phase; SCH 58251 was ineffective if present during retrieval only. No effects were detected in the Elevated Plus maze or Open Field Test. The finding that CB1R-mediated memory disruption is prevented by antagonism of adenosine A2ARs, highlights a possibility to prevent cognitive side effects when therapeutic application of CB1R drugs is desired.

  20. Oppositional Effects of Serotonin Receptors 5-HT1a, 2, and 2c in the Regulation of Adult Hippocampal Neurogenesis

    PubMed Central

    Klempin, Friederike; Babu, Harish; Tonelli, Davide De Pietri; Alarcon, Edson; Fabel, Klaus; Kempermann, Gerd

    2009-01-01

    Serotonin (5-HT) appears to play a major role in controlling adult hippocampal neurogenesis and thereby it is relevant for theories linking failing adult neurogenesis to the pathogenesis of major depression and the mechanisms of action of antidepressants. Serotonergic drugs lacked acute effects on adult neurogenesis in many studies, which suggested a surprisingly long latency phase. Here we report that the selective serotonin reuptake inhibitor fluoxetine, which has no acute effect on precursor cell proliferation, causes the well-described increase in net neurogenesis upon prolonged treatment partly by promoting the survival and maturation of new postmitotic neurons. We hypothesized that this result is the cumulative effect of several 5-HT-dependent events in the course of adult neurogenesis. Thus, we used specific agonists and antagonists to 5-HT1a, 2, and 2c receptor subtypes to analyze their impact on different developmental stages. We found that 5-HT exerts acute and opposing effects on proliferation and survival or differentiation of precursor cells by activating the diverse receptor subtypes on different stages within the neuronal lineage in vivo. This was confirmed in vitro by demonstrating that 5-HT1a receptors are involved in self-renewal of precursor cells, whereas 5-HT2 receptors effect both proliferation and promote neuronal differentiation. We propose that under acute conditions 5-HT2 effects counteract the positive proliferative effect of 5-HT1a receptor activation. However, prolonged 5-HT2c receptor activation fosters an increase in late-stage progenitor cells and early postmitotic neurons, leading to a net increase in adult neurogenesis. Our data indicate that serotonin does not show effect latency in the adult dentate gyrus. Rather, the delayed response to serotonergic drugs with respect to endpoints downstream of the immediate receptor activity is largely due to the initially antagonistic and un-balanced action of different 5-HT receptors. PMID

  1. Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism, antagonism, or inverse agonism?

    PubMed

    Meneses, Alfredo

    2002-12-01

    1. The 5-HT2 receptors subdivision into the 5-HT(2A/2B/2C) subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT2 receptors role on memory consolidation. 2. The SB-200646 (a selective 5-HT(2B/2C) receptor antagonist) and LY215840 (a nonselective 5-HT(2/7) receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP). 3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (+/-)-2.5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT2A receptor antagonist) and to a LY215840 high dose. 4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine: while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs. 5. It is suggested that 5-HT(2B/2C) receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT2 receptors remains unclear at this time. 6. Notably, the 5-HT2 receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreasedcholinergic, glutamatergic, and/or serotonergic neurotransmission.

  2. Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids.

    PubMed

    Carriba, Paulina; Ortiz, Oskar; Patkar, Kshitij; Justinova, Zuzana; Stroik, Jessica; Themann, Andrea; Müller, Christa; Woods, Anima S; Hope, Bruce T; Ciruela, Francisco; Casadó, Vicent; Canela, Enric I; Lluis, Carme; Goldberg, Steven R; Moratalla, Rosario; Franco, Rafael; Ferré, Sergi

    2007-11-01

    The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.

  3. mGlu5, Dopamine D2 and Adenosine A2A Receptors in L-DOPA-induced Dyskinesias

    PubMed Central

    Morin, Nicolas; Morissette, Marc; Grégoire, Laurent; Di Paolo, Thérèse

    2016-01-01

    Patients with Parkinson’s disease (PD) receiving L-3,4-dihydroxyphenylalanine (L-DOPA, the gold-standard treatment for this disease) frequently develop abnormal involuntary movements, termed L-DOPA-induced dyskinesias (LID). Glutamate overactivity is well documented in PD and LID. An approach to manage LID is to add to L-DOPA specific agents to reduce dyskinesias such as metabotropic glutamate receptor (mGlu receptor) drugs. This article reviews the contribution of mGlu type 5 (mGlu5) receptors in animal models of PD. Several mGlu5 negative allosteric modulators acutely attenuate LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys and 6-hydroxydopamine(6-OHDA)-lesioned rats. Chronic administration of mGlu5 negative allosteric modulators to MPTP monkeys and 6-OHDA rats also attenuates LID while maintaining the anti-parkinsonian effect of L-DOPA. Radioligand autoradiography shows an elevation of striatal mGlu5 receptors of dyskinetic L-DOPA-treated MPTP monkeys but not in those without LID. The brain molecular correlates of the long-term effect of mGlu5 negative allosteric modulators treatments with L-DOPA attenuating development of LID was shown to extend beyond mGlu5 receptors with normalization of glutamate activity in the basal ganglia of L-DOPA-induced changes of NMDA, AMPA, mGlu2/3 receptors and VGlut2 transporter. In the basal ganglia, mGlu5 receptor negative allosteric modulators also normalize the L-DOPA-induced changes of dopamine D2 receptors, their associated signaling proteins (ERK1/2 and Akt/GSK3β) and neuropeptides (preproenkephalin, preprodynorphin) as well as the adenosine A2A receptors expression. These results show in animal models of PD reduction of LID with mGlu5 negative allosteric modulation associated with normalization of glutamate, dopamine and adenosine receptors suggesting a functional link of these receptors in chronic treatment with L-DOPA. PMID:26639458

  4. mGlu5, Dopamine D2 and Adenosine A2A Receptors in L-DOPA-induced Dyskinesias.

    PubMed

    Morin, Nicolas; Morissette, Marc; Grégoire, Laurent; Di Paolo, Thérèse

    2016-01-01

    Patients with Parkinson's disease (PD) receiving L-3,4-dihydroxyphenylalanine (L-DOPA, the gold-standard treatment for this disease) frequently develop abnormal involuntary movements, termed L-DOPA-induced dyskinesias (LID). Glutamate overactivity is well documented in PD and LID. An approach to manage LID is to add to L-DOPA specific agents to reduce dyskinesias such as metabotropic glutamate receptor (mGlu receptor) drugs. This article reviews the contribution of mGlu type 5 (mGlu5) receptors in animal models of PD. Several mGlu5 negative allosteric modulators acutely attenuate LID in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) monkeys and 6-hydroxydopamine(6-OHDA)-lesioned rats. Chronic administration of mGlu5 negative allosteric modulators to MPTP monkeys and 6-OHDA rats also attenuates LID while maintaining the antiparkinsonian effect of L-DOPA. Radioligand autoradiography shows an elevation of striatal mGlu5 receptors of dyskinetic L-DOPA-treated MPTP monkeys but not in those without LID. The brain molecular correlates of the long-term effect of mGlu5 negative allosteric modulators treatments with L-DOPA attenuating development of LID was shown to extend beyond mGlu5 receptors with normalization of glutamate activity in the basal ganglia of L-DOPA-induced changes of NMDA, AMPA, mGlu2/3 receptors and VGlut2 transporter. In the basal ganglia, mGlu5 receptor negative allosteric modulators also normalize the L-DOPA-induced changes of dopamine D2receptors, their associated signaling proteins (ERK1/2 and Akt/GSK3β) and neuropeptides (preproenkephalin, preprodynorphin) as well as the adenosine A2A receptors expression. These results show in animal models of PD reduction of LID with mGlu5 negative allosteric modulation associated with normalization of glutamate, dopamine and adenosine receptors suggesting a functional link of these receptors in chronic treatment with L-DOPA.

  5. Modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the A(2B) receptor.

    PubMed

    Ben Addi, Abduelhakem; Lefort, Anne; Hua, Xiaoyang; Libert, Frédérick; Communi, Didier; Ledent, Catherine; Macours, Pascale; Tilley, Stephen L; Boeynaems, Jean-Marie; Robaye, Bernard

    2008-06-01

    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.

  6. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function.

    PubMed

    Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Valadas, Jorge S; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E; Hamdane, Malika; Outeiro, Tiago F; Bader, Michael; Meijsing, Sebastiaan H; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V

    2016-08-11

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer's disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer's and age-related cognitive impairments may rely on its ability to modulate GR actions.

  7. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function

    PubMed Central

    Batalha, Vânia L.; Ferreira, Diana G.; Coelho, Joana E.; Valadas, Jorge S.; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E.; Hamdane, Malika; Outeiro, Tiago F.; Bader, Michael; Meijsing, Sebastiaan H.; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V.

    2016-01-01

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer’s disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer’s and age-related cognitive impairments may rely on its ability to modulate GR actions. PMID:27510168

  8. Adenosine A2A receptor deletion affects social behaviors and anxiety in mice: Involvement of anterior cingulate cortex and amygdala.

    PubMed

    López-Cruz, Laura; Carbó-Gas, Maria; Pardo, Marta; Bayarri, Pilar; Valverde, Olga; Ledent, Catherine; Salamone, John D; Correa, Mercè

    2017-03-15

    Blockade of adenosine A2A receptors can potentiate motivation to work for natural reinforcers such as food. Conspecific interaction is a potent natural reinforcer in social animals that can be manifested as preference for social exploration versus other sources of novel stimulation. Deficiencies in this type of motivated behavior (social withdrawal) have been seen in several pathologies such as autism and depression. However, the role of A2A receptors in motivation for social interaction has not been widely explored. Social interaction paradigms evaluate the natural preference of animals for exploring other conspecifics, and the ability to differentiate between familiar versus novel ones. Anxiety is one of the factors that can induce avoidance of social interaction. In the present study, adenosine A2A knockout (A2AKO) and wild-type (WT) mice were assessed for social and anxiety-related behaviors. c-Fos immunoreactivity was evaluated as a measure of neuronal activation in brain areas involved in different aspects of motivation and emotional processes. Although A2AKO mice showed an anxious profile, they displayed higher levels of sociability and were less sensitive to social novelty. WT mice displayed a typical pattern of social recognition 24h later, but not A2AKO mice, which explored equally both conspecifics. There were no differences between strains in aggressiveness, perseverance or social odor preferences. c-Fos immunoreactivity in A2AKO mice was higher in anterior cingulate and amygdala compared to WT mice. Thus, A2A receptors appear to be potential targets for the improvement of pathologies related to social function.

  9. Binding mode similarity measures for ranking of docking poses: a case study on the adenosine A2A receptor

    NASA Astrophysics Data System (ADS)

    Anighoro, Andrew; Bajorath, Jürgen

    2016-06-01

    We report an investigation designed to explore alternative approaches for ranking of docking poses in the search for antagonists of the adenosine A2A receptor, an attractive target for structure-based virtual screening. Calculation of 3D similarity of docking poses to crystallographic ligand(s) as well as similarity of receptor-ligand interaction patterns was consistently superior to conventional scoring functions for prioritizing antagonists over decoys. Moreover, the use of crystallographic antagonists and agonists, a core fragment of an antagonist, and a model of an agonist placed into the binding site of an antagonist-bound form of the receptor resulted in a significant early enrichment of antagonists in compound rankings. Taken together, these findings showed that the use of binding modes of agonists and/or antagonists, even if they were only approximate, for similarity assessment of docking poses or comparison of interaction patterns increased the odds of identifying new active compounds over conventional scoring.

  10. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    PubMed

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar

    2013-01-30

    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  11. Cocaine self-administration differentially affects allosteric A2A-D2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder.

    PubMed

    Pintsuk, Julia; Borroto-Escuela, Dasiel O; Pomierny, Bartosz; Wydra, Karolina; Zaniewska, Magdalena; Filip, Malgorzata; Fuxe, Kjell

    2016-05-01

    In the current study behavioral and biochemical experiments were performed to study changes in the allosteric A2AR-D2R interactions in the ventral and dorsal striatum after cocaine self-administration versus corresponding yoked saline control. By using ex vivo [(3)H]-raclopride/quinpirole competition experiments, the effects of the A2AR agonist CGS 21680 (100 nM) on the KiH and KiL values of the D2-like receptor (D2-likeR) were determined. One major result was a significant reduction in the D2-likeR agonist high affinity state observed with CGS 21680 after cocaine self-administration in the ventral striatum compared with the yoked saline group. The results therefore support the hypothesis that A2AR agonists can at least in part counteract the motivational actions of cocaine. This action is mediated via the D2-likeR by targeting the A2AR protomer of A2AR-D2-like R heteroreceptor complexes in the ventral striatum, which leads to the reduction of D2-likeR protomer recognition through the allosteric receptor-receptor interaction. In contrast, in the dorsal striatum the CGS 21680-induced antagonistic modulation in the D2-likeR agonist high affinity state was abolished after cocaine self-administration versus the yoked saline group probably due to a local dysfunction/disruption of the A2AR-D2-like R heteroreceptor complexes. Such a change in the dorsal striatum in cocaine self-administration can contribute to the development of either locomotor sensitization, habit-forming learning and/or the compulsive drug seeking by enhanced D2-likeR protomer signaling. Potential differences in the composition and stoichiometry of the A2AR-D2R heteroreceptor complexes, including differential recruitment of sigma 1 receptor, in the ventral and dorsal striatum may explain the differential regional changes observed in the A2A-D2-likeR interactions after cocaine self-administration.

  12. The orphan nuclear receptor NR4A2 is part of a p53–microRNA-34 network

    PubMed Central

    Beard, Jordan A.; Tenga, Alexa; Hills, Justin; Hoyer, Jessica D.; Cherian, Milu T.; Wang, Yong-Dong; Chen, Taosheng

    2016-01-01

    Nuclear receptor subfamily 4 group A member 2 (NR4A2) is an orphan nuclear receptor that is over-expressed in cancer and promotes cell proliferation, migration, transformation, and chemoresistance. Increased expression and function of NR4A2 have been attributed to various signaling pathways, but little is known about microRNA (miRNA) regulation of NR4A2 in cancer. To investigate the posttranscriptional regulation of NR4A2, we used a 3′ untranslated region (UTR) reporter screen and identified miR-34 as a putative regulator of NR4A2. By using computer predictions, we identified and confirmed an miRNA recognition element in the 3′ UTR of NR4A2 that was responsible for miR-34–mediated suppression. We next demonstrated that overexpression of exogenous miR-34 or activation of the p53 pathway, which regulates endogenous miR-34 expression, decreased NR4A2 expression. Consistent with previous reports, overexpression of NR4A2 blocked the induction of p53 target genes, including mir-34a. This was a phenotypic effect, as NR4A2 overexpression could rescue cells from p53-induced inhibition of proliferation. In summary, our results are the first characterization of a cancer-related miRNA capable of regulating NR4A2 and suggest a network and possible feedback mechanism involving p53, miR-34, and NR4A2. PMID:27121375

  13. Structure and dynamics of AMPA receptor GluA2 in resting, pre-open and desensitized states

    PubMed Central

    Dürr, Katharina L.; Chen, Lei; Stein, Richard A.; De Zorzi, Rita; MihaelaFolea, I.; Walz, Thomas; Mchaourab, Hassane S.; Gouaux, Eric

    2014-01-01

    Summary Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs in the nervous system, little is known about the structures and dynamics of intact receptors in distinct functional states. Here we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with the partial agonists and a positive allosteric modulator and in a desensitized/closed state in complex with FW alone. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryo-electron microscopy studies. We show how agonist binding modulates the conformation of the ligand binding domain 'layer' of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation and desensitization in AMPA iGluRs. PMID:25109876

  14. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    NASA Astrophysics Data System (ADS)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  15. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  16. Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor.

    PubMed

    Boilard, Eric; Rouault, Morgane; Surrel, Fanny; Le Calvez, Catherine; Bezzine, Sofiane; Singer, Alan; Gelb, Michael H; Lambeau, Gérard

    2006-11-07

    Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.

  17. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    PubMed

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  18. Adenosine A2A Receptor Blockade Prevents Rotenone-Induced Motor Impairment in a Rat Model of Parkinsonism

    PubMed Central

    Fathalla, Ahmed M.; Soliman, Amira M.; Ali, Mohamed H.; Moustafa, Ahmed A.

    2016-01-01

    Pharmacological studies implicate the blockade of adenosine receptorsas an effective strategy for reducing Parkinson’s disease (PD) symptoms. The objective of this study is to elucidate the possible protective effects of ZM241385 and 8-cyclopentyl-1, 3-dipropylxanthine, two selective A2A and A1 receptor antagonists, on a rotenone rat model of PD. Rats were split into four groups: vehicle control (1 ml/kg/48 h), rotenone (1.5 mg/kg/48 h, s.c.), ZM241385 (3.3 mg/kg/day, i.p) and 8-cyclopentyl-1, 3-dipropylxanthine (5 mg/kg/day, i.p). After that, animals were subjected to behavioral (stride length and grid walking) and biochemical (measuring concentration of dopamine levels using high performance liquid chromatography, HPLC). In the rotenone group, rats displayed a reduced motor activity and disturbed movement coordination in the behavioral tests and a decreased dopamine concentration as foundby HPLC. The effect of rotenone was partially prevented in the ZM241385 group, but not with 8-cyclopentyl-1,3-dipropylxanthine administration. The administration of ZM241385 improved motor function and movement coordination (partial increase of stride length and partial decrease in the number of foot slips) and an increase in dopamine concentration in the rotenone-injected rats. However, the 8-cyclopentyl-1,3-dipropylxanthine and rotenone groups were not significantly different. These results indicate that selective A2A receptor blockade by ZM241385, but not A1 receptor blockadeby 8-cyclopentyl-1,3-dipropylxanthine, may treat PD motor symptoms. This reinforces the potential use of A2A receptor antagonists as a treatment strategy for PD patients. PMID:26973484

  19. Involvement of central beta2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats.

    PubMed

    Malinowska, B; Zakrzeska, A; Kurz, C M; Göthert, M; Kwolek, G; Wielgat, P; Braszko, J J; Schlicker, E

    2010-04-01

    Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A(2) TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and beta(2)-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5-3 micromol/kg, i.v.) or its stable analogue methanandamide (0.75 micromol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 mumol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB(1) and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 mumol/kg each), and/or SQ 29548 (1 mumol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 mumol per animal i.c.v.) and by the thromboxane A(2) synthesis inhibitor furegrelate i.c.v. (1.8 micromol per animal). ICI 118551, MK-801 (1 micromol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central beta(2)-adrenergic, NMDA and thromboxane A(2) receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.

  20. The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center.

    PubMed

    Abbott, Robert K; Silva, Murillo; Labuda, Jasmine; Thayer, Molly; Cain, Derek W; Philbrook, Phaethon; Sethumadhavan, Shalini; Hatfield, Stephen; Ohta, Akio; Sitkovsky, Michail

    2017-01-27

    T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

  1. Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor.

    PubMed

    Eini, Hadar; Frishman, Valeria; Yulzari, Robert; Kachko, Leonid; Lewis, Eli C; Chaimovitz, Cidio; Douvdevani, Amos

    2015-11-01

    Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development.

  2. Histamine H3 Receptor Activation Counteracts Adenosine A2A Receptor-Mediated Enhancement of Depolarization-Evoked [3H]-GABA Release from Rat Globus Pallidus Synaptosomes

    PubMed Central

    2014-01-01

    High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [3H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [3H]-GABA release induced by high K+ (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-3H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K+-evoked [3H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K+-induced [3H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections. PMID:24884070

  3. Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

    PubMed

    Kecskés, Miklós; Kumar, T Santhosh; Yoo, Lena; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-08-15

    Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

  4. Low-dose aspirin in prevention of miscarriage in women with unexplained or autoimmune related recurrent miscarriage: effect on prostacyclin and thromboxane A2 production.

    PubMed

    Tulppala, M; Marttunen, M; Söderstrom-Anttila, V; Foudila, T; Ailus, K; Palosuo, T; Ylikorkala, O

    1997-07-01

    Early pregnancies in women with a history of recurrent spontaneous abortion (RSA) are accompanied by a deficiency in vasodilatory and anti-aggregatory prostacyclin (PGI2) and/or overproduction of its endogenous antagonist thromboxane A2 (TXA2). We evaluated the effect of a low-dose aspirin (LDA) on PGI2 and TXA2 production and on pregnancy outcome in RSA women with and without detectable anticardiolipin antibodies (ACA). Of 82 RSA women studied, 66 became pregnant, and of them, 33 (six with elevated and 27 with normal ACA concentrations) were randomized to receive LDA (50 mg/day) and 33 (six with elevated and 27 with normal ACA concentrations) to receive placebo (PLA) from a mean of 6.6 days after the missed period to delivery. Treatment with LDA inhibited platelet TXA2 production similarly in RSA women with and without detectable ACA and with continuing pregnancies (7.0 +/- 0.7 ng/ml, LDA group versus 254.5 +/- 37.8 ng/ml, PLA group, mean +/- SEM, P < 0.0001) or miscarrying pregnancies (13.8 +/- 3.8 ng/ml compared with 233.6 +/- 59.8 ng/ml, P < 0.0001 respectively). Furthermore, LDA decreased urinary excretion of the TXA2 metabolite (2,3-dinor-TXB2) both in pregnancies which went to term (6.1 +/- 0.6 ng/mmol creatinine, LDA group versus 19.3 +/- 3.0 ng/mmol creatinine, PLA group, P < 0.0001) or again ended in miscarriage (4.7 +/- 0.8 ng/mmol creatinine versus 17.3 +/- 4.4 ng/mmol creatinine, P < 0.0001 respectively), but did not affect the excretion of the prostacyclin metabolite (2,3-dinor-6-keto-PGF1alpha). Early pregnancy ultrasound examination revealed a living fetus in 58 women. Of these, seven in the LDA group (23.3%, four with elevated and three with normal ACA concentrations) and five in the PLA group (17.9%, two with elevated and three with normal ACA concentrations; not significant) experienced a miscarriage. All infants were healthy, and the frequency of growth retardation was similar in both groups (13.0%). One woman in the LDA group (4.3%) and three

  5. The role of the plexin-A2 receptor in Sema3A and Sema3B signal transduction.

    PubMed

    Sabag, Adi D; Smolkin, Tatyana; Mumblat, Yelena; Ueffing, Marius; Kessler, Ofra; Gloeckner, Christian Johannes; Neufeld, Gera

    2014-12-15

    Class 3 semaphorins are anti-angiogenic and anti-tumorigenic guidance factors that bind to neuropilins, which, in turn, associate with class A plexins to transduce semaphorin signals. To study the role of the plexin-A2 receptor in semaphorin signaling, we silenced its expression in endothelial cells and in glioblastoma cells. The silencing did not affect Sema3A signaling, which depended on neuropilin-1, plexin-A1 and plexin-A4, but completely abolished Sema3B signaling, which also required plexin-A4 and one of the two neuropilins. Interestingly, overexpression of plexin-A2 in plexin-A1- or plexin-A4-silenced cells restored responses to both semaphorins, although it nullified their ability to differentiate between them, suggesting that, when overexpressed, plexin-A2 can functionally replace other class A plexins. By contrast, although plexin-A4 overexpression restored Sema3A signaling in plexin-A1-silenced cells, it failed to restore Sema3B signaling in plexin-A2-silenced cells. It follows that the identity of plexins in functional semaphorin receptors can be flexible depending on their expression level. Our results suggest that changes in the expression of plexins induced by microenvironmental cues can trigger differential responses of different populations of migrating cells to encountered gradients of semaphorins.

  6. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  7. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  8. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  9. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-06-09

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs.

  10. Up-regulation of striatal adenosine A(2A) receptors with iron deficiency in rats: effects on locomotion and cortico-striatal neurotransmission.

    PubMed

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-07-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A(2A) receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A(2A) receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A(2A) receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A(2A) receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A(2A) receptors was found in rats fed during 3 weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A(2A) receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A(2A) receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A(2A) receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A(2A) receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS.

  11. A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice.

    PubMed

    Belikoff, Bryan G; Hatfield, Stephen; Georgiev, Peter; Ohta, Akio; Lukashev, Dmitriy; Buras, Jon A; Remick, Daniel G; Sitkovsky, Michail

    2011-02-15

    Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

  12. Dipyridamole attenuates ischemia reperfusion induced acute kidney injury through adenosinergic A1 and A2A receptor agonism in rats.

    PubMed

    Puri, Nikkita; Mohey, Vinita; Singh, Manjinder; Kaur, Tajpreet; Pathak, Devendra; Buttar, Harpal Singh; Singh, Amrit Pal

    2016-04-01

    Dipyridamole (DYP) is an anti-platelet agent with marked vasodilator, anti-oxidant, and anti-inflammatory activity. The present study investigated the role of adenosine receptors in DYP-mediated protection against ischemia reperfusion-induced acute kidney injury (AKI) in rats. The rats were subjected to bilateral renal ischemia for 40 min followed by reperfusion for 24 h. The renal damage induced by ischemia reperfusion injury (IRI) was assessed by measuring creatinine clearance, blood urea nitrogen, uric acid, plasma potassium, fractional excretion of sodium, and microproteinuria in rats. The oxidative stress in renal tissues was assessed by quantification of thiobarbituric acid-reactive substances, superoxide anion generation, and reduced glutathione level. The hematoxylin-eosin staining was carried out to observe histopathological changes in renal tissues. DYP (10 and 30 mg/kg, intraperitoneal, i.p.) was administered 30 min before subjecting the rats to renal IRI. In separate groups, caffeine (50 mg/kg, i.p.), an adenosinergic A1 and A2A receptor antagonist was administered with and without DYP treatment before subjecting the rats to renal IRI. The ischemia reperfusion-induced AKI was demonstrated by significant changes in serum as well as urinary parameters, enhanced oxidative stress, and histopathological changes in renal tissues. The administration of DYP demonstrated protection against AKI. The prior treatment with caffeine abolished DYP-mediated reno-protection suggesting role of A1 and A2A adenosine receptors in DYP-mediated reno-protection in rats. It is concluded that adenosine receptors find their definite involvement in DYP-mediated anti-oxidative and reno-protective effect against ischemia reperfusion-induced AKI.

  13. Selecting an optimal number of binding site waters to improve virtual screening enrichments against the adenosine A2A receptor.

    PubMed

    Lenselink, Eelke B; Beuming, Thijs; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

    2014-06-23

    A major challenge in structure-based virtual screening (VS) involves the treatment of explicit water molecules during docking in order to improve the enrichment of active compounds over decoys. Here we have investigated this in the context of the adenosine A2A receptor, where water molecules have previously been shown to be important for achieving high enrichment rates with docking, and where the positions of some binding site waters are known from a high-resolution crystal structure. The effect of these waters (both their presence and orientations) on VS enrichment was assessed using a carefully curated set of 299 high affinity A2A antagonists and 17,337 decoys. We show that including certain crystal waters greatly improves VS enrichment and that optimization of water hydrogen positions is needed in order to achieve the best results. We also show that waters derived from a molecular dynamics simulation - without any knowledge of crystallographic waters - can improve enrichments to a similar degree as the crystallographic waters, which makes this strategy applicable to structures without experimental knowledge of water positions. Finally, we used decision trees to select an ensemble of structures with different water molecule positions and orientations that outperforms any single structure with water molecules. The approach presented here is validated against independent test sets of A2A receptor antagonists and decoys from the literature. In general, this water optimization strategy could be applied to any target with waters-mediated protein-ligand interactions.

  14. Role of adenosine A2A receptor signaling in the nicotine-evoked attenuation of reflex cardiac sympathetic control.

    PubMed

    El-Mas, Mahmoud M; El-Gowilly, Sahar M; Fouda, Mohamed A; Saad, Evan I

    2011-08-01

    Baroreflex dysfunction contributes to increased cardiovascular risk in cigarette smokers. Given the importance of adenosinergic pathways in baroreflex control, the hypothesis was tested that defective central adenosinergic modulation of cardiac autonomic activity mediates the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate (HR) to increases or decreases in blood pressure (BP) evoked by i.v. doses (1-16μg/kg) of phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious rats; slopes of the curves were taken as measures of baroreflex sensitivity (BRS). Nicotine (25 and 100μg/kg i.v.) dose-dependently reduced BRS(SNP) in contrast to no effect on BRS(PE). BRS(SNP) was also attenuated after intracisternal (i.c.) administration of nicotine. Similar reductions in BRS(SNP) were observed in rats pretreated with atropine or propranolol. The combined treatment with nicotine and atropine produced additive inhibitory effects on BRS, an effect that was not demonstrated upon concurrent exposure to nicotine and propranolol. BRS(SNP) was reduced in preparations treated with i.c. 8-phenyltheophylline (8-PT, nonselective adenosine receptor antagonist), 8-(3-Chlorostyryl) caffeine (CSC, A(2A) antagonist), or VUF5574 (A(3) antagonist). In contrast, BRS(SNP) was preserved after blockade of A(1) (DPCPX) or A(2B) (alloxazine) receptors or inhibition of adenosine uptake by dipyridamole. CSC or 8-PT abrogated the BRS(SNP) depressant effect of nicotine whereas other adenosinergic antagonists were without effect. Together, nicotine preferentially impairs reflex tachycardia via disruption of adenosine A(2A) receptor-mediated facilitation of reflex cardiac sympathoexcitation. Clinically, the attenuation by nicotine of compensatory sympathoexcitation may be detrimental in conditions such as hypothalamic defense response, posture changes, and ventricular rhythms.

  15. Adenosine A2A receptors and uric acid mediate protective effects of inosine against TNBS-induced colitis in rats.

    PubMed

    Rahimian, Reza; Fakhfouri, Gohar; Daneshmand, Ali; Mohammadi, Hamed; Bahremand, Arash; Rasouli, Mohammad Reza; Mousavizadeh, Kazem; Dehpour, Ahmad Reza

    2010-12-15

    Inflammatory bowel disease comprises chronic recurrent inflammation of gastrointestinal tract. This study was conducted to investigate inosine, a potent immunomodulator, in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced chronic model of experimental colitis, and contribution of adenosine A(2A) receptors and the metabolite uric acid as possible underlying mechanisms. Experimental colitis was rendered in rats by a single colonic administration of 10 mg of TNBS. Inosine, potassium oxonate (a hepatic uricase inhibitor), SCH-442416 (a selective adenosine A(2A) receptor antagonist), inosine+potassium oxonate, or inosine+SCH-442416 were given twice daily for 7 successive days. At the end of experiment, macroscopic and histopathologic scores, colonic malondialdehyde (MDA), Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-1beta (IL-1β) levels, and myeloperoxidase (MPO) activity were assessed. Plasma uric acid level was measured throughout the experiment. Both macroscopic and histological features of colonic injury were markedly ameliorated by either inosine, oxonate or inosine+oxonate. Likewise, the elevated amounts of MPO and MDA abated as well as those of TNF-α and IL-1β (P<0.05). SCH-442416 partially reversed the effect of inosine on theses markers, while inosine+oxonate showed a higher degree of protection than each treatment alone (P<.0.05). No significant difference was observed between TNBS and SCH-442416 groups. Uric acid levels were significantly higher in inosine or oxonate groups compared to control. Inosine+oxonate resulted in an even more elvelated uric acid level than each treatment alone (P<0.05). Inosine elicits notable anti-inflammatory effects on TNBS-induced colitis in rats. Uric acid and adenosine A(2A) receptors contribute to these salutary properties.

  16. Sinomenine Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice via Adenosine A2A Receptor Signaling

    PubMed Central

    Li, Jun; Zhao, Li; He, Xie; Zeng, Yi-Jun; Dai, Shuang-Shuang

    2013-01-01

    Sinomenine (SIN) is a bioactive alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, which is widely used in the clinical treatment of rheumatoid arthritis (RA). However, its role in acute lung injury (ALI) is unclear. In this study, we investigate the role of SIN in lipopolysaccharide (LPS)-induced ALI in mice. After ALI, lung water content and histological signs of pulmonary injury were attenuated, whereas the PaO2/FIO2 (P/F) ratios were elevated significantly in the mice pretreated with SIN. Additionally, SIN markedly inhibited inflammatory cytokine TNF-α and IL-1β expression levels as well as neutrophil infiltration in the lung tissues of the mice. Microarray analysis and real-time PCR showed that SIN treatment upregulated adenosine A2A receptor (A2AR) expression, and the protective effect of SIN was abolished in A2AR knockout mice. Further investigation in isolated mouse neutrophils confirmed the upregulation of A2AR by SIN and showed that A2AR-cAMP-PKA signaling was involved in the anti-inflammatory effect of SIN. Taken together, these findings demonstrate an A2AR-associated anti-inflammatory effect and the protective role of SIN in ALI, which suggests a potential novel approach to treat ALI. PMID:23555007

  17. Sinomenine protects against lipopolysaccharide-induced acute lung injury in mice via adenosine A(2A) receptor signaling.

    PubMed

    Li, Jun; Zhao, Li; He, Xie; Zeng, Yi-Jun; Dai, Shuang-Shuang

    2013-01-01

    Sinomenine (SIN) is a bioactive alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, which is widely used in the clinical treatment of rheumatoid arthritis (RA). However, its role in acute lung injury (ALI) is unclear. In this study, we investigate the role of SIN in lipopolysaccharide (LPS)-induced ALI in mice. After ALI, lung water content and histological signs of pulmonary injury were attenuated, whereas the PaO2/FIO2 (P/F) ratios were elevated significantly in the mice pretreated with SIN. Additionally, SIN markedly inhibited inflammatory cytokine TNF-α and IL-1β expression levels as well as neutrophil infiltration in the lung tissues of the mice. Microarray analysis and real-time PCR showed that SIN treatment upregulated adenosine A(2A) receptor (A(2A)R) expression, and the protective effect of SIN was abolished in A(2A)R knockout mice. Further investigation in isolated mouse neutrophils confirmed the upregulation of A(2A)R by SIN and showed that A(2A)R-cAMP-PKA signaling was involved in the anti-inflammatory effect of SIN. Taken together, these findings demonstrate an A(2A)R-associated anti-inflammatory effect and the protective role of SIN in ALI, which suggests a potential novel approach to treat ALI.

  18. IFN-γ prevents adenosine receptor (A2bR) upregulation to sustain the macrophage activation response

    PubMed Central

    Cohen, Heather B.; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M.

    2015-01-01

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. Here, we demonstrate that following TLR stimulation, macrophages up regulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This up-regulation of A2bR leads to the induction of a macrophage with an immunoregulatory phenotype and the down regulation of inflammation. IFN-γ priming of macrophages, selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNFα and IL-12 in response to TLR ligation. The pharmacological inhibition or the genetic deletion of the A2bR results in a hyper-inflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense, by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the anti-microbial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  19. Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE).

    PubMed

    Barros-Barbosa, Aurora R; Ferreirinha, Fátima; Oliveira, Ângela; Mendes, Marina; Lobo, M Graça; Santos, Agostinho; Rangel, Rui; Pelletier, Julie; Sévigny, Jean; Cordeiro, J Miguel; Correia-de-Sá, Paulo

    2016-12-01

    Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.

  20. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies

    DTIC Science & Technology

    2015-02-01

    functional and histological changes asso ciated with diabetic nephropathy in wild type diabetic mice but not in the A2AAR−/− diabetic mice (Awad et al...the beginning of streptozotocin induced diabetes at the age of eight weeks. This treatment , previously demonstrated to increase free adenosine levels in...and it was not affected by ABT 702 treatment Blood glucose levels were higher in diabetic mice compared with non diabetic groups and they were not

  1. A2B adenosine receptors stimulate IL-6 production in primary murine microglia through p38 MAPK kinase pathway.

    PubMed

    Merighi, Stefania; Bencivenni, Serena; Vincenzi, Fabrizio; Varani, Katia; Borea, Pier Andrea; Gessi, Stefania

    2017-03-01

    The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of proinflammatory mediators implicated in the pathogenesis of neuronal diseases. Adenosine is an ubiquitous autacoid regulating several microglia functions through four receptor subtypes named A1, A2A, A2B and A3 (ARs), that represent good targets to suppress inflammation occurring in CNS. Here we investigated the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A2BAR agonist 2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide (BAY60-6583) stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and PKC delta (PKC-δ) the IL-6 increase due to A2BAR activation was strongly reduced, whilst it was not affected by the inhibitor of adenylyl cyclase (AC). Investigation of cellular signalling involved in the A2BAR effect revealed that only the inhibitor of p38 mitogen activated protein kinase (MAPK) was able to block the agonist's effect on IL-6 secretion, whilst inhibitors of pERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY60-6583 was A2BAR-dependent, through a pathway affecting PLC, PKC-ε and PKC-δ but not AC, in both normoxia and hypoxia. Finally, BAY60-6583 increased microglial cell proliferation involving A2BAR, PLC, PKC-ε, PKC-δ and p38 signalling. In conclusion, A2BARs activation increased IL-6 secretion and cell proliferation in murine primary microglial cells, through PLC, PKC-ε, PKC-δ and p38 pathways, thus suggesting their involvement in microglial activation and neuroinflammation.

  2. Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.

    PubMed

    Mayer, Gaétan; Poirier, Steve; Seidah, Nabil G

    2008-11-14

    The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

  3. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    SciTech Connect

    Bolado-Carrancio, A.; Riancho, J.A.; Sainz, J.; Rodríguez-Rey, J.C.

    2014-04-04

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.

  4. Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression*

    PubMed Central

    Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Nurbek; Mambetsariev, Bolot; Salgia, Ravi; Singleton, Patrick A.

    2014-01-01

    Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression. PMID:25023279

  5. Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

    PubMed

    Freitag, Christine M; Agelopoulos, Konstantin; Huy, Ellen; Rothermundt, Matthias; Krakowitzky, Petra; Meyer, Jobst; Deckert, Jürgen; von Gontard, Alexander; Hohoff, Christa

    2010-01-01

    Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

  6. Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine.

    PubMed

    van der Walt, M M; Terre'Blanche, G

    2017-01-05

    Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 μM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 μM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

  7. Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor.

    PubMed

    Piirainen, Henni; Taura, Jaume; Kursula, Petri; Ciruela, Francisco; Jaakola, Veli-Pekka

    2017-04-01

    Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A2A receptor (A2AR), has an exceptionally long intracellular C terminus (A2AR-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A2AR and the role of Ca(2+) in this process. First, we studied the A2AR-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A2AR-ct through its distal calmodulin-like domain in a Ca(2+)-independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the A2AR-calmodulin/Ca(2+) complex. Importantly, calmodulin displaced α-actinin 1 from the A2AR-ct in a Ca(2+)-dependent fashion, disrupting the A2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca(2+) on A2AR internalization in living cells, a function operated by the A2AR-α-actinin 1 complex. Interestingly, while Ca(2+) influx did not affect constitutive A2AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A2AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A2AR with calmodulin and α-actinin 1 is fine-tuned by Ca(2+), a fact that might power agonist-mediated receptor internalization and function.

  8. Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A 2A receptors in striatal projection neurons.

    PubMed

    Hernández-González, O; Hernández-Flores, T; Prieto, G A; Pérez-Burgos, A; Arias-García, M A; Galarraga, E; Bargas, J

    2014-01-01

    D(1)- and D(2)-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A(1)-type receptors are located in both neuron classes, and adenosine A(2A)-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca(2+)-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D(1)-type receptors increase, while D(2)-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, CaV1 Ca(2+)-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A(1)- and A(2A)-receptors have not been compared observing their actions on Ca(2+)-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca(2+)-currents by A(1)-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A(1)- and A2A-receptors. We demonstrate that A(1)-type receptors reduced Ca(2+)-currents in all SPNs tested. However, A(2A)-type receptors enhanced Ca(2+)-currents only in half tested neurons. Intriguingly, to observe the actions of A(2A)-type receptors, occupation of A(1)-type receptors had to occur first. However, A(1)-receptors decreased Ca(V)2 Ca(2+)-currents, while A(2A)-type receptors enhanced current through Ca(V)1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.

  9. Expression of the TPα and TPβ isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential

    PubMed Central

    Mulvaney, Eamon P.; Shilling, Christine; Eivers, Sarah B.; Perry, Antoinette S.; Bjartell, Anders; Kay, Elaine W.; Watson, R. William; Kinsella, B. Therese

    2016-01-01

    The prostanoid thromboxane (TX) A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPβ, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown. This study evaluated expression of the TPα and TPβ isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPβ was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPβ expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPβ in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPβ, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPβ expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPβ, or a combination of TPα/TPβ, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence. PMID:27689401

  10. Ex vivo Perfusion with Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs after Circulatory Death

    PubMed Central

    Stone, Mathew L.; Sharma, Ashish K.; Mas, Valeria. R.; Gehrau, Ricardo C.; Mulloy, Daniel P.; Zhao, Yunge; Lau, Christine L.; Kron, Irving L.; Laubach, Victor E.

    2015-01-01

    Background Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs prior to transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Methods Mice underwent euthanasia and 60 min warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 min). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 min (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP+ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Results EVLP significantly improved lung function versus CSP, which was further, significantly improved by EVLP+ATL1223. Lung edema, cytokines and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP+ATL1223. Gene array analysis revealed differential expression of 1,594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6 and IL-17 signaling. Several pathways were uniquely regulated by EVLP+ATL1223 including the downregulation of genes involved in IL-1 signaling such as ADCY9, ECSIT, IRAK1, MAPK12 and TOLLIP. Conclusion EVLP modulates pro-inflammatory genes and reduces pulmonary dysfunction, edema and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs. PMID:26262504

  11. Genetic blockade of adenosine A2A receptors induces cognitive impairments and anatomical changes related to psychotic symptoms in mice.

    PubMed

    Moscoso-Castro, Maria; Gracia-Rubio, Irene; Ciruela, Francisco; Valverde, Olga

    2016-07-01

    Schizophrenia is a chronic severe mental disorder with a presumed neurodevelopmental origin, and no effective treatment. Schizophrenia is a multifactorial disease with genetic, environmental and neurochemical etiology. The main theories on the pathophysiology of this disorder include alterations in dopaminergic and glutamatergic neurotransmission in limbic and cortical areas of the brain. Early hypotheses also suggested that nucleoside adenosine is a putative affected neurotransmitter system, and clinical evidence suggests that adenosine adjuvants improve treatment outcomes, especially in poorly responsive patients. Hence, it is important to elucidate the role of the neuromodulator adenosine in the pathophysiology of schizophrenia. A2A adenosine receptor (A2AR) subtypes are expressed in brain areas controlling motivational responses and cognition, including striatum, and in lower levels in hippocampus and cerebral cortex. The aim of this study was to characterize A2AR knockout (KO) mice with complete and specific inactivation of A2AR, as an animal model for schizophrenia. We performed behavioral, anatomical and neurochemical studies to assess psychotic-like symptoms in adult male and female KO and wild-type (WT) littermates. Our results show impairments in inhibitory responses and sensory gating in A2AR KO animals. Hyperlocomotion induced by d-amphetamine and MK-801 was reduced in KO animals when compared to WT littermates. Moreover, A2AR KO animals show motor disturbances, social and cognitive alterations. Finally, behavioral impairments were associated with enlargement of brain lateral ventricles and decreased BDNF levels in the hippocampus. These data highlight the role of adenosine in the pathophysiology of schizophrenia and provide new possibilities for the therapeutic management of schizophrenia.

  12. Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor.

    PubMed

    Granata, Francescopaolo; Petraroli, Angelica; Boilard, Eric; Bezzine, Sofiane; Bollinger, James; Del Vecchio, Luigi; Gelb, Michael H; Lambeau, Gerard; Marone, Gianni; Triggiani, Massimo

    2005-01-01

    Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

  13. Formation of 8-iso-PGF(2alpha) and thromboxane A(2) by stimulation with several activators of phospholipase A(2) in the isolated human umbilical vein.

    PubMed

    Sametz, W; Hummer, K; Butter, M; Wintersteiger, R; Juan, H

    2000-09-01

    We investigated the effects of the phospholipase A(2) (PLA(2)) activators calcium ionophore A 23187, hydrogen peroxide (H(2)O(2)), bradykinin (BK), histamine and noradrenaline (NA) on the 8-iso-prostaglandin (PG)F(2alpha) formation in the isolated human umbilical vein and the isolated rabbit ear. For comparison, the influence of these substances on the thromboxane A(2) (TXA(2)) release was also investigated. The release of total (esterified as well as free) 8-iso-PGF(2alpha), free 8-iso-PGF(2alpha) and TXB(2), the stable metabolite of TXA(2), was determined by specific enzyme immunoassays. The results show that bolus injections of 5.4 mmol H(2)O(2), 30 nmol A 23187, 10 nmol BK, 50 nmol histamine and 20 nmol NA caused an increased release of total 8-iso-PGF(2alpha) in the umbilical vein and the rabbit ear. A perfusion with H(2)O(2) at a final concentration of 0.3 mM also increased the release of this isoprostane. Increased formation of free 8-iso-PGF(2alpha) was induced by A 23187 injection and by both modes of H(2)O(2) administration, but not by the other treatments. Bolus injections of A 23187, BK and histamine induced an increased release of TXB(2) in both organs. Both modes of H(2)O(2) administration and NA showed no releasing effects. In conclusion, our results show that the substances used are able to stimulate the formation of 8-iso-PGF(2alpha) concurrently with the release of PGs. This effect might be of pathophysiological relevance in inflammatory and cardiovascular diseases in which an enhanced release of free radicals, BK, histamine or NA play an important role.

  14. Persistent reduction of cocaine seeking by pharmacological manipulation of adenosine A1 and A2A receptors during extinction training in rats

    PubMed Central

    O’Neill, Casey E.; Hobson, Benjamin D.; Levis, Sophia C.; Bachtell, Ryan K.

    2014-01-01

    Rationale Adenosine receptor stimulation and blockade has been shown to modulate a variety of cocaine related behaviors. Objectives These studies identify the direct effects of adenosine receptor stimulation on cocaine seeking during extinction training and the persistent effects on subsequent reinstatement to cocaine seeking. Methods Rats self-administered cocaine on a fixed-ratio 1 schedule in daily sessions over 3 weeks. Following 1 week withdrawal, the direct effects of adenosine receptor modulation were tested by administering the adenosine A1 receptor agonist, CPA (0.03 mg/kg and 0.1 mg/kg), the adenosine A2A agonist, CGS 21680 (0.03 mg/kg and 0.1 mg/kg), the presynaptic adenosine A2A receptor antagonist, SCH 442416 (0.3 mg/kg, 1 mg/kg, and 3 mg/kg), or vehicle prior to each of 6 daily extinction sessions. The persistent effects of adenosine receptor modulation during extinction training were subsequently tested on reinstatement to cocaine seeking induced by cues, cocaine, and the dopamine D2 receptor agonist, quinpirole. Results All doses of CPA and CGS 21680 impaired initial extinction responding, however only CPA treatment during extinction produced persistent impairment in subsequent cocaine- and quinpirole-induced seeking. Dissociating CPA treatment from extinction did not alter extinction responding or subsequent reinstatement. Administration of SCH 442416 had no direct effects on extinction responding, but produced dose-dependent persistent impairment of cocaine- and quinpirole-induced seeking. Conclusions These findings demonstrate that adenosine A1 or A2A receptor stimulation directly impair extinction responding. Interestingly, adenosine A1 receptor stimulation or presynaptic adenosine A2A receptor blockade during extinction produces lasting changes in relapse susceptibility. PMID:24562064

  15. Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

    PubMed

    Sun, Kaiqi; Zhang, Yujin; Bogdanov, Mikhail V; Wu, Hongyu; Song, Anren; Li, Jessica; Dowhan, William; Idowu, Modupe; Juneja, Harinder S; Molina, Jose G; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-03-05

    Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

  16. Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

    PubMed Central

    Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

    2015-01-01

    Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

  17. Salidroside attenuates chronic hypoxia-induced pulmonary hypertension via adenosine A2a receptor related mitochondria-dependent apoptosis pathway.

    PubMed

    Huang, Xiaoying; Zou, Lizhen; Yu, Xiaoming; Chen, Mayun; Guo, Rui; Cai, Hui; Yao, Dan; Xu, Xiaomei; Chen, Yanfan; Ding, Cheng; Cai, Xueding; Wang, Liangxing

    2015-05-01

    Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Salidroside, an active ingredient isolated from Rhodiola rosea is proposed to exert protective effects against PAH. However, the function of salidroside in PAH has not been investigated systematically and the underlying mechanisms are not clear. To investigate the effects of salidroside on PAH, the mice in chronic hypoxia model of PAH were given by an increasing concentration of salidroside (0, 16 mg/kg, 32 mg/kg, and 64 mg/kg). After salidroside treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling were attenuated, suggesting a protective role played by salidroside in PAH. To explore the potential mechanisms, the apoptosis of PASMCs after salidroside treatment under hypoxia conditions were determined in vivo and in vitro, and also the mitochondria-dependent apoptosis factors, Bax, Bcl-2, cytochrome C, and caspase 9 were examined. The results revealed that salidroside reversed hypoxia-induced cell apoptosis resistance at least partially via a mitochondria-dependent pathway. In addition, salidroside upregulated the expression of adenosine A2a receptor (A2aR) in lung tissues of mice and in PASMCs in vitro after hypoxia exposure. Combined the evidence above, we conclude that salidroside can attenuate chronic hypoxia-induced PAH by promoting PASMCs apoptosis via an A2aR related mitochondria dependent pathway.

  18. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2016-01-01

    The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology. PMID:27043281

  19. Chronic oral administration of MPEP, an antagonist of mGlu5 receptor, during gestation and lactation alters mGlu5 and A2A receptors in maternal and neonatal brain.

    PubMed

    López-Zapata, Antonio; León-Navarro, David Agustín; Crespo, María; Albasanz, José Luis; Martín, Mairena

    2017-03-06

    Antidepressant and anxiolytic drugs are widely consumed even by pregnant and lactating women. The metabotropic glutamate receptor 5 (mGlu5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) exerts antidepressant- and anxiolytic-like actions. Given that treatment for anxiety and depression use to be prolonged in time, it is conceivable a possible modulation of metabotropic glutamate receptors (mGlu receptors) after prolonged MPEP exposure, which could also modify adenosine A2A receptors (A2AR) since functional cross-talk between them has been reported. Here we report that MPEP crosses placental barrier and reaches neonatal brain through maternal milk using LC-MS/MS methods. Therefore, we analyzed mGlu receptors, mainly mGlu5, and A2AR in both maternal and fetal brain after chronic maternal consumption of MPEP during gestation and/or lactation using radioligand binding, Western-blotting, real-time PCR and phospholipase C (PLC) activity assays. In maternal brain, chronic MPEP consumption caused a significant loss of mGlu, including mGlu5, and A2AR receptors level in plasma membrane. PLC activity assays showed that mGlu5 signaling pathway was desensitized. No variations on mRNA level coding A2AR, A1R and mGlu5 were found after MPEP treatments. In female neonatal brain, maternal consumption of MPEP caused a significant increase in mGlu, including mGlu5, and A2AR receptors level. Neither mGlu receptors nor A2AR were modified in male neonatal brain after maternal MPEP intake. Finally, neither molecular nor behavioral changes (anxiety- and depression-like behavior) were observed in 3-month-old female offspring. In summary, mGlu5 and A2AR are altered in both maternal and female neonatal brain after chronic maternal consumption of MPEP during gestation and/or lactation.

  20. Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses

    PubMed Central

    Wedemeyer, Carolina; Zorrilla de San Martín, Javier; Ballestero, Jimena; Gómez-Casati, María Eugenia; Torbidoni, Ana Vanesa; Fuchs, Paul A.; Bettler, Bernhard; Elgoyhen, Ana Belén

    2013-01-01

    The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses. PMID:24068816

  1. Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

    PubMed Central

    Correia-de-Sá, P.; Ribeiro, J. A.

    1994-01-01

    1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8004402

  2. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

  3. The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors.

    PubMed

    Seibt, Benjamin F; Schiedel, Anke C; Thimm, Dominik; Hinz, Sonja; Sherbiny, Farag F; Müller, Christa E

    2013-05-01

    The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A2A and A2B by exchanging the complete EL2 of the human A2BAR for the EL2 of the A2AAR. Furthermore, single amino acid residues (Asp148(45.27), Ser149(45.28), Thr151(45.30), Glu164(45.43), Ser165(45.44), and Val169(45.48)) in the EL2 of the A2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A2A-selective agonist CGS21680, while being completely inactive at A2BARs, showed high affinity for the mutant A2B(EL2-A2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A2B(EL2-A2A) as compared to the wt AR. Thus, the EL2 of the A2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.

  4. Wound healing effects of noni (Morinda citrifolia L.) leaves: a mechanism involving its PDGF/A2A receptor ligand binding and promotion of wound closure.

    PubMed

    Palu, Afa; Su, Chen; Zhou, Bing-Nan; West, Brett; Jensen, Jarakae

    2010-10-01

    Morinda citrifolia L. (Rubiaceae) commonly known as noni, has been used in Polynesia by traditional healers for the treatment of cuts, bruises and wounds. Our objective was to investigate the wound-healing mechanisms of the noni leaf. The investigations of its wound-healing mechanisms were carried out using fresh noni leaf juice (NLJ), noni leaf ethanol extract (NLEE) and its methanol (MFEE) and hexane (HFEE) fractions on the PDGF and A(2A) receptors in vitro and topically in mice. Fresh noni leaf juice showed significant affinity to PDGF receptors, and displayed 166% binding inhibition of the ligand binding to its receptors, while at the same concentration, it only had 7% inhibition of the ligand binding to the A(2A) receptors. NLEE, HFEE and MFEE showed significant affinity to A(2A) receptors, concentration dependently, with IC(50) values of 34.1, 42.9 and 86.7 μg/mL, respectively. However, MFEE significantly increased wound closure and reduced the half closure time in mice with a CT(50) of 5.4 ± 0.2 days compared with control (p < 0.05). These results suggest that noni leaf significantly accelerated wound healing in mice via its ligand binding to the PDGF and A(2A) receptors as its probable mechanisms of wound-healing and also support its traditional usage for wound-healing in Polynesia.

  5. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions.

    PubMed

    Cao, Ruyin; Rossetti, Giulia; Bauer, Andreas; CarIoni, Paolo

    2015-01-01

    Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD) study of the human adenosine receptor type 2A (hA(2A)R) in complex with caffeine--a system of high neuro-pharmacological relevance--within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system), often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA(2A)R and possibly other GPCRs.

  6. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions

    PubMed Central

    Cao, Ruyin; Rossetti, Giulia; Bauer, Andreas; CarIoni, Paolo

    2015-01-01

    Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD) study of the human adenosine receptor type 2A (hA2AR) in complex with caffeine—a system of high neuro-pharmacological relevance—within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system), often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA2AR and possibly other GPCRs. PMID:25992797

  7. Effect of adenosine A(2A) receptor antagonists on L-DOPA-induced hydroxyl radical formation in rat striatum.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna; Kowalska, Magdalena; Kamińska, Katarzyna

    2009-02-01

    A(2A) adenosine receptor antagonists have been proposed as a new therapy for Parkinson's disease (PD). Since oxidative stress plays an important role in the pathogenesis of PD, we studied the effect of the selective A(2A) adenosine receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on L: -3,4-dihydroxyphenylalanine (L: -DOPA)-induced hydroxyl radical generation using in vivo microdialysis in the striatum of freely moving rats. L: -DOPA (100 mg/kg; in the presence of benserazide, 50 mg/kg) given acutely or repeatedly for 14 days generated a high level of hydroxyl radicals, measured by HPLC with electrochemical detection, as the product of their reaction with p-hydroxybenzoic acid (PBA). CSC (1 mg/kg) and ZM 241385 (3 mg/kg) decreased haloperidol (0.5 mg/kg)-induced catalepsy, while at low doses of 0.1 and 0.3 mg/kg, respectively, they did not display an effect. CSC (1 and 5 mg/kg) and ZM 241385 (3 and 9 mg/kg) given acutely, or CSC (1 mg/kg) and ZM 241385 (3 mg/kg) given repeatedly, increased the production of hydroxyl radicals in dialysates from rat striatum. Both acute and repeated administration of CSC (0.1 and 1 mg/kg) and ZM 241385 (3 mg/kg) decreased L: -DOPA-induced generation of hydroxyl radicals. However, a high single dose of either CSC (5 mg/kg) and ZM 241385 (9 mg/kg) markedly potentiated the effect of L: -DOPA on hydroxyl radical production. The increase in hydroxyl radical production by acute and chronic injection of CSC and ZM 241385 may be related to the increased release of dopamine (DA) and its metabolism in striatal dialysates. Similarly, increased DA release following a single high dose of CSC or ZM 241385 appears to be responsible for augmentation of L: -DOPA-induced hydroxyl radical formation. Conversely, the inhibition of L: -DOPA-induced production of hydroxyl radical by single and repeated low doses of CSC or repeated low doses of ZM

  8. Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2.

    PubMed

    Krintel, Christian; Francotte, Pierre; Pickering, Darryl S; Juknaitė, Lina; Pøhlsgaard, Jacob; Olsen, Lars; Frydenvang, Karla; Goffin, Eric; Pirotte, Bernard; Kastrup, Jette S

    2016-06-07

    The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from -4.9 (2) and -7.5 (3) to -6.2 (4) and -14.5 (5), but also a less favorable binding entropy (-TΔS, kcal/mol) from -2.3 (2) and -1.3 (3) to -0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, μM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 μM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

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

    PubMed Central

    Alencar, Allan K N; Pereira, Sharlene L; Montagnoli, Tadeu L; Maia, Rodolfo C; Kümmerle, Arthur E; Landgraf, Sharon S; Caruso-Neves, Celso; Ferraz, Emanuelle B; Tesch, Roberta; Nascimento, José H M; de Sant'Anna, Carlos M R; Fraga, Carlos A M; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2013-01-01

    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, A2A, A3) and of docking with A2A 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 A2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A2A receptor agonist, 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 A2A receptors. PMID:23530610

  10. Prostaglandin A2 Enhances Cellular Insulin Sensitivity via a Mechanism that Involves the Orphan Nuclear Receptor NR4A3

    PubMed Central

    Zhu, X.; Walton, R. G.; Tian, L.; Luo, N.; Ho, S-R.; Fu, Y.; Garvey, W. T.

    2014-01-01

    We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin’s ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3. PMID:23104421

  11. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2

    PubMed Central

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-01-01

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2. PMID:27973457

  12. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2.

    PubMed

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-12-12

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2.

  13. Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

    PubMed Central

    Tognolini, Massimiliano; Incerti, Matteo; Mohamed, Iftiin Hassan; Giorgio, Carmine; Russo, Simonetta; Bruni, Renato; Lelli, Barbara; Bracci, Luisa; Noberini, Roberta; Pasquale, Elena B.; Barocelli, Elisabetta; Vicini, Paola; Mor, Marco

    2012-01-01

    The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions. PMID:22529030

  14. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    PubMed

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena

    2015-08-01

    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  15. Plexin-A4-dependent retrograde semaphorin 3A signalling regulates the dendritic localization of GluA2-containing AMPA receptors.

    PubMed

    Yamashita, Naoya; Usui, Hiroshi; Nakamura, Fumio; Chen, Sandy; Sasaki, Yukio; Hida, Tomonobu; Suto, Fumikazu; Taniguchi, Masahiko; Takei, Kohtaro; Goshima, Yoshio

    2014-03-06

    The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.

  16. Plasticity in the contribution of T cell receptor variable region residues to binding of peptide-HLA-A2 complexes

    PubMed Central

    Smith, Sheena N.; Sommermeyer, Daniel; Piepenbrink, Kurt H.; Blevins, Sydney J.; Bernhard, Helga; Uckert, Wolfgang; Baker, Brian M.; Kranz, David M.

    2013-01-01

    One hypothesis to account for MHC-restriction by T cell receptors (TCRs) holds that there are several evolutionary-conserved residues in TCR variable regions that contact MHC. While this ‘germline-codon’ hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key ‘germline codon” residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. Using single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type ‘germline codon’ residues, but several alternative residues could also accommodate binding and hence, MHC-restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e. MHC-restriction), there is significant plasticity in requirements for particular side-chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs. PMID:23954306

  17. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

    PubMed

    Slee, Deborah H; Moorjani, Manisha; Zhang, Xiaohu; Lin, Emily; Lanier, Marion C; Chen, Yongsheng; Rueter, Jaimie K; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Joswig, Tanya; Santos, Mark; Gross, Raymond S; Williams, John P; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Wen, Jenny; O'Brien, Zhihong; Petroski, Robert; Saunders, John

    2008-03-27

    Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.

  18. sPLA2 IB induces human podocyte apoptosis via the M-type phospholipase A2 receptor.

    PubMed

    Pan, Yangbin; Wan, Jianxin; Liu, Yipeng; Yang, Qian; Liang, Wei; Singhal, Pravin C; Saleem, Moin A; Ding, Guohua

    2014-10-22

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content.

  19. Neuroprotective potential of adenosine A2A and cannabinoid CB1 receptor antagonists in an animal model of Parkinson disease.

    PubMed

    Cerri, Silvia; Levandis, Giovanna; Ambrosi, Giulia; Montepeloso, Elena; Antoninetti, Gian Filippo; Franco, Rafael; Lanciego, José Luis; Baqi, Younis; Müller, Christa E; Pinna, Annalisa; Blandini, Fabio; Armentero, Marie Therese

    2014-05-01

    The development of nondopaminergic therapeutic strategies that may improve motor and nonmotor deficits, while possibly slowing down the neurodegenerative process and associated neuroinflammation,is a primary goal of Parkinson disease (PD) research. We investigated the neuroprotective and anti-inflammatory potential of combined and single treatment with adenosine A2A and cannabinoid CB1 receptor antagonists MSX-3 and rimonabant, respectively, in a rodent model of PD. Rats bearing a unilateral intrastriatal 6-hydroxydopamine lesion were treated chronically with MSX-3 (0.5or 1 mg/kg/d) and rimonabant (0.1 mg/kg/d) given as monotherapy or combined. The effects of the treatments to counteract dopaminergic cell death and neuroinflammation were assessed by immunohistochemistry for tyrosine hydroxylase and glial cell markers, respectively. Both rimonabant and MSX-3 (1 mg/kg/d) promoted dopaminergic neuron survival in the substantia nigra pars compacta (SNc) when given alone; this effect was weakened when the compounds were combined. Glial activation was not significantly affected by MSX-3 (1 mg/kg/d), whereas rimonabant seemed to increase astrocyte cell density in the SNc. Our findings demonstrate the neuroprotective potential of single treatments and suggest that glial cells might be involved in this protective effect. The results also indicate that the neuroprotective potential of combined therapy may not necessarily reflect or promote single-drug effects and point out that special care should be taken when considering multidrug therapies in PD.

  20. Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs)

    PubMed Central

    Orr, Katy; Buckley, Niamh E.; Haddock, Paula; James, Colin; Parent, Jean-Luc; McQuaid, Stephen; Mullan, Paul B.

    2016-01-01

    Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets. We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -‘good’ outcome no relapse > 3 years; ‘poor’ outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in ‘good’ outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression. We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments. PMID:27487152

  1. Involvement of adenosine A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats.

    PubMed

    Justinova, Zuzana; Ferre, Sergi; Segal, Pavan N; Antoniou, Katerina; Solinas, Marcello; Pappas, Lara A; Highkin, Jena L; Hockemeyer, Jorg; Munzar, Patrik; Goldberg, Steven R

    2003-12-01

    Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the

  2. A novel conjugated agent between dopamine and an A2A adenosine receptor antagonist as a potential anti-Parkinson multitarget approach.

    PubMed

    Dalpiaz, Alessandro; Cacciari, Barbara; Vicentini, Chiara Beatrice; Bortolotti, Fabrizio; Spalluto, Giampiero; Federico, Stephanie; Pavan, Barbara; Vincenzi, Fabrizio; Borea, Pier Andrea; Varani, Katia

    2012-03-05

    We propose a potential antiparkinsonian prodrug DP-L-A(2A)ANT (2) obtained by amidic conjugation of dopamine (1) via a succinic spacer to a new triazolo-triazine A(2A) adenosine receptor (AR) antagonist A(2A)ANT (3). The affinity of 2 and its hydrolysis products-1, 3, dopamine-linker DP-L (4) and A(2A)ANT-linker L-A(2A)ANT (5)-was evaluated for hA(1), hA(2A), hA(2B) and hA(3) ARs and rat striatum A(2A)ARs or D(2) receptors. The hydrolysis patterns of 2, 4 and 5 and the stabilities of 1 and 3 were evaluated by HPLC analysis in human whole blood and rat brain homogenates. High hA(2A) affinity was shown by compounds 2 (K(i) = 7.32 ± 0.65 nM), 3 (K(i) = 35 ± 3 nM) and 5 (K(i) = 72 ± 5 nM), whose affinity values were similar in rat striatum. These compounds were not able to change dopamine affinity for D(2) receptors but counteracted the CGS 21680-induced reduction of dopamine affinity. DP-L (4) was inactive on adenosine and dopaminergic receptors. As for stability studies, compounds 4 and 5 were not degraded in incubation media. In human blood, the prodrug 2 was hydrolyzed (half-life = 2.73 ± 0.23 h) mainly on the amidic bound coupling the A(2A)ANT (3), whereas in rat brain homogenates the prodrug 2 was hydrolyzed (half-life > eight hours) exclusively on the amidic bound coupling dopamine, allowing its controlled release and increasing its poor stability as characterized by half-life = 22.5 ± 1.5 min.

  3. Caffeine consumption prevents memory impairment, neuronal damage, and adenosine A2A receptors upregulation in the hippocampus of a rat model of sporadic dementia.

    PubMed

    Espinosa, Janaína; Rocha, Andreia; Nunes, Fernanda; Costa, Marcelo S; Schein, Vanessa; Kazlauckas, Vanessa; Kalinine, Eduardo; Souza, Diogo O; Cunha, Rodrigo A; Porciúncula, Lisiane O

    2013-01-01

    Intracerebroventricular (icv) streptozotocin (STZ) administration induces pathological and behavioral alterations similar to those observed in Alzheimer's disease (AD) and is thus considered an experimental model of sporadic AD. Since caffeine (an adenosine receptor antagonist) and selective antagonists of adenosine A2A receptors modify the course of memory impairment in different amyloid-β-based experimental models of AD, we now tested the impact of caffeine on STZ-induced dementia and associated neurodegeneration in the hippocampus as well as on the expression and density of adenosine receptors. Adult male rats received a bilateral infusion of saline or STZ (3 mg/kg, icv), which triggered memory deficits after four weeks, as gauged by impaired object recognition memory. This was accompanied by a reduced NeuN immunoreactivity in the hippocampal CA1 region and an increased expression and density of adenosine A2A receptors (A2AR), but not A1R, in the hippocampus. Caffeine consumption (1 g/L in the drinking water starting 2 weeks before the STZ challenge) prevented the STZ-induced memory impairment and neurodegeneration as well as the upregulation of A2AR. These findings provide the first demonstration that caffeine prevents sporadic dementia and implicate the control of central A2AR as its likely mechanism of action.

  4. Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5'-flanking region.

    PubMed

    Yoshinari, Kouichi; Yoda, Noriaki; Toriyabe, Takayoshi; Yamazoe, Yasushi

    2010-01-15

    Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5'-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from -461 to -554 and from -18089 to -21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around -520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor alpha heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

  5. Differential Expression of Adenosine A1 and A2A Receptors After Upper Cervical (C2) Spinal Cord Hemisection in Adult Rats

    PubMed Central

    Petrov, Theodor; Kreipke, Christian; Alilain, Warren; Nantwi, Kwaku D

    2007-01-01

    Background: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3–C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. Objective: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. Methods: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3× daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3× daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. Results: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. Conclusion: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection–induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline

  6. Initial Evaluation of a Novel Adenosine A2A Receptor Ligand, (11)C-Preladenant, in Healthy Human Subjects.

    PubMed

    Sakata, Muneyuki; Ishibashi, Kenji; Imai, Masamichi; Wagatsuma, Kei; Ishii, Kenji; Zhou, Xiaoyun; de Vries, Erik F; Elsinga, Philip H; Ishiwata, Kiichi; Toyohara, Jun

    2017-03-09

    (11)C-Preladenant is a novel selective antagonist for mapping of cerebral adenosine A2A receptors (A2ARs) by positron emission tomography (PET). This is a first-in-human study to examine the safety, radiation dosimetry, and brain imaging of (11)C-preladenant in healthy human subjects. Methods: Dynamic (11)C-preladenant PET scans (90 min) were performed in 5 healthy male subjects. During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined. For anatomic coregistration, T1-weighted magnetic resonance imaging was performed. The total distribution volume (VT) was estimated using one- and two-tissue compartment models (1T and 2T, respectively). Distribution volume ratio (DVR) was calculated from VT of target and reference region, and obtained with a non-invasive Logan graphical reference tissue method (LGRM) (t* = 30 min). The applicability of a shortened protocol as an alternative to the 90 min PET scan was investigated. Tracer biodistribution and dosimetry were determined in 3 healthy male subjects, using serial whole-body PET scan acquired over 2 h post (11)C-preladenant injection. Results: There were no serious adverse events in any of the subjects throughout the study period. (11)C-Preladenat readily entered the brain, with a peak uptake in the putamen and head of the caudate nucleus 30-40 min after tracer injection. Other brain regions showed rapid clearance of radioactivity. The regional distribution of (11)C-preladenant was consistent with known A2AR densities in the brain. At pseudoequilibrium (reached at 40 min after injection), stable target-to-cerebellar cortex ratios of around 3.8-10.0 were obtained. The 2T fit better than the 1T in the low-density A2AR regions. In contrast, there were no significant differences between 1T and 2T in the high A2AR density regions. DVRs in putamen and head of the caudate nucleus were around 3.8-10.3 when estimated using a LGRM with cerebellum as the

  7. A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration In Vitro and Prostate Cancer Metastasis In Vivo

    PubMed Central

    Guo, Hong; Miao, Hui; Tochtrop, Gregory P.; Hsieh, Jer-Tsong; Page, Phillip; Liu, Lili; Lindner, Daniel J.; Acharya, Chayan; MacKerell, Alexander D.; Ficker, Eckhard; Song, Jianxing; Wang, Bingcheng

    2012-01-01

    During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo. PMID:22916121

  8. Arginine vasotocin V1a2 receptor and GnRH-I co-localize in preoptic neurons of the sex changing grouper, Epinephelus adscensionis.

    PubMed

    Kline, Richard J; Holt, G Joan; Khan, Izhar A

    2016-01-01

    The arginine vasotocin/vasopressin (AVT/AVP) and gonadotropin releasing hormone (GnRH) systems are known to control sexual behaviors and reproduction, respectively, in different vertebrate groups. However, a direct functional connection between these two neuroendocrine systems has not been demonstrated for any vertebrate species. Therefore, the objective of this research was to test the hypothesis that AVT acts on the GnRH system via an AVT V1a receptor in a sex changing grouper species, the rock hind, Epinephelus adscensionis. AVT V1a2 receptors were co-localized with GnRH-I on neurons in the preoptic anterior hypothalamus identifying a structural linkage between the AVT system and GnRH-I. Transcripts for avt, gnrh-I, and two AVT receptor subtypes (v1a1 and v1a2) were isolated and characterized for E. adscensionis and their expression was measured in males and females by q-RT-PCR. Translation of V1a-type cDNA sequences revealed two distinct forms of the AVT V1a receptor in E. adscensionis brain similar to those reported for other species. The observation of significantly higher gnrh-I mRNA in the POA+H of rock hind males as compared to females suggests differential regulation of the gnrh-I transcripts in the two sexes of this protogynous species. In male E. adscensionis, but not in females, a negative relationship was seen between plasma 11-ketotestosterone (11-KT) and the v1a1 receptor mRNA levels in the POA+H, while a positive trend was observed between 11-KT and v1a2 receptor mRNA levels, indicating that these receptor forms may be differentially regulated.

  9. Synthesis and pharmacological evaluation of dual acting ligands targeting the adenosine A2A and dopamine D2 receptors for the potential treatment of Parkinson's disease.

    PubMed

    Jörg, Manuela; May, Lauren T; Mak, Frankie S; Lee, Kiew Ching K; Miller, Neil D; Scammells, Peter J; Capuano, Ben

    2015-01-22

    A relatively new strategy in drug discovery is the development of dual acting ligands. These molecules are potentially able to interact at two orthosteric binding sites of a heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher affinity, enhanced or modified physiological response, and reduced reliance on multiple drug administration regimens. In this study, we have successfully synthesized a series of classical heterobivalent ligands as well as a series of more integrated and "drug-like" dual acting molecules, incorporating ropinirole as a dopamine D2 receptor agonist and ZM 241385 as an adenosine A2A receptor antagonist. The best compounds of our series maintained the potency of the original pharmacophores at both receptors (adenosine A2A and dopamine D2). In addition, the integrated dual acting ligands also showed promising results in preliminary blood-brain barrier permeability tests, whereas the classical heterobivalent ligands are potentially more suited as pharmacological tools.

  10. Adenosine A2A receptor antagonism and genetic deletion attenuate the effects of dopamine D2 antagonism on effort-based decision making in mice.

    PubMed

    Pardo, M; Lopez-Cruz, L; Valverde, O; Ledent, C; Baqi, Y; Müller, C E; Salamone, J D; Correa, M

    2012-04-01

    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 D(2) 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 A(2A) receptor but not the A(1) receptor, since the A(2A) antagonist MSX-3, but not the A(1) 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 A(2A) receptor knockout mice were resistant to the effects of haloperidol on effort-related choice in the maze. These results indicate that DA D(2) and adenosine A(2A) receptors interact to regulate effort-related decision making and effort expenditure in mice.

  11. Adenosine A2A receptor agonist prevents cardiac remodeling and dysfunction in spontaneously hypertensive male rats after myocardial infarction

    PubMed Central

    da Silva, Jaqueline S; Gabriel-Costa, Daniele; Sudo, Roberto T; Wang, Hao; Groban, Leanne; Ferraz, Emanuele B; Nascimento, José Hamilton M; Fraga, Carlos Alberto M; Barreiro, Eliezer J; Zapata-Sudo, Gisele

    2017-01-01

    Background This work evaluated the hypothesis that 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), an agonist of adenosine A2A receptor, could be beneficial for preventing cardiac dysfunction due to hypertension associated with myocardial infarction (MI). Methods Male spontaneously hypertensive rats (SHR) were randomly divided into four groups (six animals per group): sham-operation (SHR-Sham), and myocardial infarction rats (SHR-MI) were treated orally either with vehicle or LASSBio-294 (10 and 20 mg.kg−1.d−1) for 4 weeks. Echocardiography and in vivo hemodynamic parameters measured left ventricle (LV) structure and function. Exercise tolerance was evaluated using a treadmill test. Cardiac remodeling was accessed by LV collagen deposition and tumor necrosis factor α expression. Results Early mitral inflow velocity was significantly reduced in the SHR-MI group, and there was significant recovery in a dose-dependent manner after treatment with LASSBio-294. Exercise intolerance observed in the SHR-MI group was prevented by 10 mg.kg−1.d−1 of LASS-Bio-294, and exercise tolerance exceeded that of the SHR-Sham group at 20 mg.kg−1.d−1. LV end-diastolic pressure increased after MI, and this was prevented by 10 and 20 mg.kg−1.d−1 of LASSBio-294. Sarcoplasmic reticulum Ca2+ ATPase levels were restored in a dose-dependent manner after treatment with LASSBio-294. Fibrosis and inflammatory processes were also counteracted by LASSBio-294, with reductions in LV collagen deposition and tumor necrosis factor α expression. Conclusion In summary, oral administration of LASSBio-294 after MI in a dose-dependent manner prevented the development of cardiac dysfunction, demonstrating this compound’s potential as an alternative treatment for heart failure in the setting of ischemic heart disease with superimposed chronic hypertension. PMID:28293100

  12. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

  13. Anti-phospholipase A2 receptor antibody levels at diagnosis predicts spontaneous remission of idiopathic membranous nephropathy

    PubMed Central

    Jullien, Perrine; Seitz Polski, Barbara; Maillard, Nicolas; Thibaudin, Damien; Laurent, Blandine; Ollier, Edouard; Alamartine, Eric; Lambeau, Gérard

    2017-01-01

    Abstract Background: The diagnostic role of circulating anti-phospholipase A2 receptor antibodies (anti-PLA2R Abs) is now well recognized in idiopathic membranous nephropathy (iMN). These Abs could also be interesting as predictors of clinical outcome. In this study, we explored the prognostic value of anti-PLA2R Abs measured in a cohort of iMN patients, with a special focus on their ability to detect patients achieving spontaneous remission. Methods: All adult patients with biopsy-proven iMN diagnosed between 1978 and 2007 were retrospectively screened in our centre. Using a validated enzyme-linked immunosorbent assay, levels of anti-PLA2R Abs were measured from serum samples obtained at the time of renal biopsy and stored at −80°C until processing. Clinical data on disease activity, treatments and outcomes were collected by reviewing patients’ medical records. The association between anti-PLA2R Ab titres and clinical activity/outcome was assessed by Cox proportional hazard and Kaplan–Meier methods. Results: In this retrospective study, 68 patients were included in the final analysis (median follow-up of 81 months). No significant association was found between anti-PLA2R Ab titres at diagnosis with baseline proteinuria, baseline estimated glomerular filtration rate or chronic kidney disease progression. Spontaneous remission was observed in 22% of patients. Ab titres were significantly and gradually correlated in a dose–response manner with the likelihood of spontaneous remission. Conclusions: While Ab titres measured at diagnosis were not found to predict the activity of iMN, evaluation of anti-PLA2R Ab titres might prove useful in the early identification of patients likely to achieve spontaneous remission.

  14. Understanding the Functional Plasticity in Neural Networks of the Basal Ganglia in Cocaine Use Disorder: A Role for Allosteric Receptor-Receptor Interactions in A2A-D2 Heteroreceptor Complexes

    PubMed Central

    Borroto-Escuela, Dasiel O.; Wydra, Karolina; Pintsuk, Julia; Narvaez, Manuel; Corrales, Fidel; Zaniewska, Magdalena; Agnati, Luigi F.; Franco, Rafael; Tanganelli, Sergio; Filip, Malgorzata

    2016-01-01

    Our hypothesis is that allosteric receptor-receptor interactions in homo- and heteroreceptor complexes may form the molecular basis of learning and memory. This principle is illustrated by showing how cocaine abuse can alter the adenosine A2AR-dopamine D2R heterocomplexes and their receptor-receptor interactions and hereby induce neural plasticity in the basal ganglia. Studies with A2AR ligands using cocaine self-administration procedures indicate that antagonistic allosteric A2AR-D2R heterocomplexes of the ventral striatopallidal GABA antireward pathway play a significant role in reducing cocaine induced reward, motivation, and cocaine seeking. Anticocaine actions of A2AR agonists can also be produced at A2AR homocomplexes in these antireward neurons, actions in which are independent of D2R signaling. At the A2AR-D2R heterocomplex, they are dependent on the strength of the antagonistic allosteric A2AR-D2R interaction and the number of A2AR-D2R and A2AR-D2R-sigma1R heterocomplexes present in the ventral striatopallidal GABA neurons. It involves a differential cocaine-induced increase in sigma1Rs in the ventral versus the dorsal striatum. In contrast, the allosteric brake on the D2R protomer signaling in the A2AR-D2R heterocomplex of the dorsal striatopallidal GABA neurons is lost upon cocaine self-administration. This is potentially due to differences in composition and allosteric plasticity of these complexes versus those in the ventral striatopallidal neurons. PMID:27872762

  15. The effects of adenosine A2B receptor inhibition on VEGF and nitric oxide axis-mediated renal function in diabetic nephropathy.

    PubMed

    Patel, Leena; Thaker, Aswin

    2014-07-01

    Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide. The pathophysiologic mechanisms of diabetic nephropathy are incompletely understood but include overproduction of various growth factors and cytokines. Upregulation of vascular endothelial growth factor (VEGF) is a pathogenic event occurring in most forms of podocytopathy; however, the mechanisms that regulate this growth factor induction are not clearly identified. A2B receptors have been found to regulate VEGF expression under hypoxic environment in different tissues. One proposed hypothesis in mediating diabetic nephropathy is the modulation of VEGF-NO balance in renal tissue. We determined the role of adenosine A2B receptor in mediating VEGF overproduction and nitrite in diabetic nephropathy. The renal content of A2B receptors and VEGF was increased after 8 weeks of diabetes induction. The renal and plasma nitrite levels were also reduced in these animals. In vivo administration of A2B adenosine receptor antagonist (MRS1754) inhibited the renal over expression of VEGF and adverse renal function parameters. The antagonist administration also improved the kidney tissue nitrite levels. In conclusion, we demonstrated that VEGF induction via adenosine signaling might be the critical event in regulating VEGF-NO axis in diabetic nephropathy.

  16. Adenosine A2A receptor antagonists improve deficits in initiation of movement and sensory motor integration in the unilateral 6-hydroxydopamine rat model of Parkinson's disease.

    PubMed

    Pinna, Annalisa; Pontis, Silvia; Borsini, Franco; Morelli, Micaela

    2007-08-01

    Evidence obtained in rodent and primate models of Parkinson's disease (PD) and preliminary clinical trials, indicates that adenosine A(2A) receptor antagonists might represent a promising nondopaminergic therapeutic tool for the treatment of PD. Those studies demonstrated the ability of adenosine A(2A) receptor antagonists to potentiate l-dopa-mediated motor improvement, whereas very little is known about counteraction of specific motor deficits and on the effects of these compounds when administered alone. To this aim we evaluated the effects of different adenosine A(2A) receptor antagonists on initiation of movement deficits, gait impairment and sensory-motor deficits, induced in rats by a unilateral 6-hydroxydopamine lesion of dopaminergic nigrostriatal neurons. The following tests were used: (1) initiation time of stepping; (2) adjusting step (stepping with forelimb was measured as the forelimb was dragged laterally); (3) vibrissae-elicited forelimb placing (as index of sensory-motor integration deficits). Acute administration of the A(2A) receptor antagonists SCH 58261 (5 mg/kg i.p.) and ST 1535 (20 mg/kg i.p.) similarly to l-dopa (6 mg/kg i.p.) counteracted the impairments in the initiation time of stepping test, in the adjusting step and in the vibrissae-elicited forelimb placing induced by the lesion. The intensity of the effect was l-dopa > SCH 58261 > ST 1535. The results provide the first evidence that blockade of A(2A) receptors is effective in antagonizing specific motor deficit induced by DA neuron degeneration, such as initiation of movement and sensory-motor integration deficits, even without l-dopa combined administration.

  17. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats.

    PubMed

    Iglesias, Inmaculada; Albasanz, Jose Luis; Martín, Mairena

    2014-12-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.

  18. Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys.

    PubMed

    Justinová, Zuzana; Redhi, Godfrey H; Goldberg, Steven R; Ferré, Sergi

    2014-05-07

    Different doses of an adenosine A2A receptor antagonist MSX-3 [3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione] were found previously to either decrease or increase self-administration of cannabinoids delta-9-tetrahydrocannabinol (THC) or anandamide in squirrel monkeys. It was hypothesized that the decrease observed with a relatively low dose of MSX-3 was related to blockade of striatal presynaptic A2A receptors that modulate glutamatergic neurotransmission, whereas the increase observed with a higher dose was related to blockade of postsynaptic A2A receptors localized in striatopallidal neurons. This hypothesis was confirmed in the present study by testing the effects of the preferential presynaptic and postsynaptic A2A receptor antagonists SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] and KW-6002 [(E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], respectively, in squirrel monkeys trained to intravenously self-administer THC. SCH-442416 produced a significant shift to the right of the THC self-administration dose-response curves, consistent with antagonism of the reinforcing effects of THC. Conversely, KW-6002 produced a significant shift to the left, consistent with potentiation of the reinforcing effects of THC. These results show that selectively blocking presynaptic A2A receptors could provide a new pharmacological approach to the treatment of marijuana dependence and underscore corticostriatal glutamatergic neurotransmission as a possible main mechanism involved in the rewarding effects of THC.

  19. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  20. Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss.

    PubMed

    Saijo, Y; Sata, F; Yamada, H; Suzuki, K; Sasaki, S; Kondo, T; Gong, Y Y; Kato, E H; Shimada, S; Morikawa, M; Minakami, H; Kishi, R

    2004-10-01

    The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.

  1. A2B and A3 Adenosine Receptors Modulate Vascular Endothelial Growth Factor and Interleukin-8 Expression in Human Melanoma Cells Treated with Etoposide and Doxorubicin

    PubMed Central

    Merighi, Stefania; Simioni, Carolina; Gessi, Stefania; Varani, Katia; Mirandola, Prisco; Tabrizi, Mojgan Aghazadeh; Baraldi, Pier Giovanni; Borea, Pier Andrea

    2009-01-01

    Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF, and HIF-1 expressions in human melanoma cancer cells. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, namely, A1, A2A, A2B, and A3. We have demonstrated that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with VP-16 and doxorubicin. This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively. PMID:19794965

  2. In search of novel ligands using a structure-based approach: a case study on the adenosine A2A receptor

    NASA Astrophysics Data System (ADS)

    Lenselink, Eelke B.; Beuming, Thijs; van Veen, Corine; Massink, Arnault; Sherman, Woody; van Vlijmen, Herman W. T.; IJzerman, Adriaan P.

    2016-10-01

    In this work, we present a case study to explore the challenges associated with finding novel molecules for a receptor that has been studied in depth and has a wealth of chemical information available. Specifically, we apply a previously described protocol that incorporates explicit water molecules in the ligand binding site to prospectively screen over 2.5 million drug-like and lead-like compounds from the commercially available eMolecules database in search of novel binders to the adenosine A2A receptor (A2AAR). A total of seventy-one compounds were selected for purchase and biochemical assaying based on high ligand efficiency and high novelty (Tanimoto coefficient ≤0.25 to any A2AAR tested compound). These molecules were then tested for their affinity to the adenosine A2A receptor in a radioligand binding assay. We identified two hits that fulfilled the criterion of 50 % radioligand displacement at a concentration of 10 μM. Next we selected an additional eight novel molecules that were predicted to make a bidentate interaction with Asn2536.55, a key interacting residue in the binding pocket of the A2AAR. None of these eight molecules were found to be active. Based on these results we discuss the advantages of structure-based methods and the challenges associated with finding chemically novel molecules for well-explored targets.

  3. Creatine, similarly to ketamine, affords antidepressant-like effects in the tail suspension test via adenosine A₁ and A2A receptor activation.

    PubMed

    Cunha, Mauricio P; Pazini, Francis L; Rosa, Julia M; Ramos-Hryb, Ana B; Oliveira, Ágatha; Kaster, Manuella P; Rodrigues, Ana Lúcia S

    2015-06-01

    The benefits of creatine supplementation have been reported in a broad range of central nervous systems diseases, including depression. A previous study from our group demonstrated that creatine produces an antidepressant-like effect in the tail suspension test (TST), a predictive model of antidepressant activity. Since depression is associated with a dysfunction of the adenosinergic system, we investigated the involvement of adenosine A1 and A2A receptors in the antidepressant-like effect of creatine in the TST. The anti-immobility effect of creatine (1 mg/kg, po) or ketamine (a fast-acting antidepressant, 1 mg/kg, ip) in the TST was prevented by pretreatment of mice with caffeine (3 mg/kg, ip, nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (2 mg/kg, ip, selective adenosine A1 receptor antagonist), and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385) (1 mg/kg, ip, selective adenosine A2A receptor antagonist). In addition, the combined administration of subeffective doses of creatine and adenosine (0.1 mg/kg, ip, nonselective adenosine receptor agonist) or inosine (0.1 mg/kg, ip, nucleoside formed by the breakdown of adenosine) reduced immobility time in the TST. Moreover, the administration of subeffective doses of creatine or ketamine combined with N-6-cyclohexyladenosine (CHA) (0.05 mg/kg, ip, selective adenosine A1 receptor agonist), N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA) (0.1 mg/kg, ip, selective adenosine A2A receptor agonist), or dipyridamole (0.1 μg/mouse, icv, adenosine transporter inhibitor) produced a synergistic antidepressant-like effect in the TST. These results indicate that creatine, similarly to ketamine, exhibits antidepressant-like effect in the TST probably mediated by the activation of both adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  4. Role of adenosine A(2A) receptors in modulating synaptic functions and brain levels of BDNF: a possible key mechanism in the pathophysiology of Huntington's disease.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Chiodi, Valentina; Ferrante, Antonella; Popoli, Patrizia

    2010-09-01

    In the last few years, accumulating evidence has shown the existence of an important cross-talk between adenosine A(2A) receptors (A(2A)Rs) and brain-derived neurotrophic factor (BDNF). Not only are A(2A)Rs involved in the mechanism of transactivation of BDNF receptor TrkB, they also modulate the effect of BDNF on synaptic transmission, playing a facilitatory and permissive role. The cAMP-PKA pathway, the main transduction system operated by A(2A)Rs, is involved in such effects. Furthermore, a basal tonus of A(2A)Rs is required to allow the regulation of BDNF physiological levels in the brain, as demonstrated by the reduced protein levels measured in A(2A)Rs KO mice. The crucial role of adenosine A(2A)Rs in the maintenance of synaptic functions and BDNF levels will be reviewed here and discussed in the light of possible implications for Huntington's disease therapy, in which a joint impairment of BDNF and A(2A)Rs seems to play a pathogenetic role.

  5. A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats

    PubMed Central

    Vincenzi, Fabrizio; Padovan, Melissa; Targa, Martina; Corciulo, Carmen; Giacuzzo, Sarah; Merighi, Stefania; Gessi, Stefania; Govoni, Marcello; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    A2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A2AAR stimulation in a rat model of arthritis. We investigated A2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A2AARs in lymphocytes from RA patients. The effectiveness of A2AAR stimulation in a rat model of arthritis supported the role of A2AAR agonists as potential pharmacological treatment for RA. PMID:23326596

  6. A novel Time-resolved Fluoroimmunoassay for the quantitative detection of Antibodies against the Phospholipase A2 Receptor

    PubMed Central

    Huang, Biao; Wang, Liang; Zhang, Yi; Zhang, Jue; Zhang, Qiuhua; Xiao, Hualong; Zhou, Bin; Sun, Zhuxing; Cao, Ya-nan; Chen, Yu; Hu, Zhigang; Sheng, Huiming

    2017-01-01

    A highly sensitive time-resolved fluoroimmunoassay (TRFIA) was developed to quantify serum antibodies against the phospholipase A2 receptor (anti-PLA2R-IgG) for differential diagnosis of membranous nephropathy. Recombinant PLA2R (rPLA2R) was coated onto 96-well plates as a capture. A goat-anti-human IgG tracer was prepared with europium-chelate for detection. After bound/free separation by washing, the fluorescence counts of bound tracer were measured for quantifying serum anti-PLA2R-IgG concentration. A purified anti-PLA2R-IgG calibrator was first prepared for ensuring that consistent quantitative results could be obtained. The assay detection limit was 0.03 mg/L with linear measurement range of 0.03–340 mg/L. The intra- and inter-assay coefficients of variation (CVs) were 3.8% and 6.2%, respectively. The average serum anti-PLA2R-IgG concentration in 45 healthy volunteers, 31 IgA nephropathy, 9 lupus nephropathy, and 52 idiopathic membranous nephropathy patients was 0.53 ± 0.18 mg/L, 0.70 ± 0.41 mg/L, 1.08 ± 0.65 mg/L, and 9.00 ± 11.82 mg/L, respectively. The cut-off point for an abnormal anti-PLA2R-IgG concentration was defined as >0.89 mg/L. The positive rates in serum from patients with IgA nephropathy, lupus nephropathy, and idiopathic membranous nephropathy were 29.0%, 44.4%, and 88.5%, respectively. The availability of this quantitation method will facilitate the use of serum anti-PLA2R-IgG for diagnosing idiopathic membranous nephropathy.

  7. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    PubMed

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation.

  8. Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor

    SciTech Connect

    Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Koshiba, Takumi; Matsukura, Yasuko; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

    2005-08-01

    A 2:2 complex of highly purified GCSF receptor (Ig-CRH) with GCSF was crystallized. The crystal diffracted to 2.8 Å resolution with sufficient quality for further structure determination. The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P4{sub 1}2{sub 1}2 (or its enantiomorph P4{sub 3}2{sub 1}2), with unit-cell parameters a = b = 110.1, c = 331.8 Å. Unfortunately, this crystal form did not diffract beyond 5 Å resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF–fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 Å resolution and belonged to space group P3{sub 1}21 (or its enantiomorph P3{sub 2}21), with unit-cell parameters a = b = 134.8, c = 105.7 Å.

  9. Recombinant production and properties of binding of the full set of mouse secreted phospholipases A2 to the mouse M-type receptor.

    PubMed

    Rouault, Morgane; Le Calvez, Catherine; Boilard, Eric; Surrel, Fanny; Singer, Alan; Ghomashchi, Farideh; Bezzine, Sofiane; Scarzello, Sabine; Bollinger, James; Gelb, Michael H; Lambeau, Gérard

    2007-02-13

    To date, 12 secreted phospholipases A2 (sPLA2s) have been identified in the mouse species and divided into three structural collections (I/II/V/X, III, and XII). On the basis of their different molecular properties and tissue distributions, each sPLA2 is likely to exert distinct functions by acting as an enzyme or ligand for specific soluble proteins or receptors, among which the M-type receptor is the best-characterized target. Here, we present the properties of binding of the full set of mouse sPLA2s to the mouse M-type receptor. All enzymes have been produced in Escherichia coli or insect cells, and their properties of binding to the cloned and native M-type receptor have been determined. sPLA2s IB, IIA, IIE, IIF, and X are high-affinity ligands (K0.5 = 0.3-3 nM); sPLA2s IIC and V are low-affinity ligands (K0.5 = 30-75 nM), and sPLA2s IID, III, XIIA, and XIIB bind only very weakly or do not bind to the M-type receptor (K0.5 > 100 nM). Three exogenous parvoviral group XIII PLA2s and two fungal group XIV sPLA2s do not bind to the receptor. Together, these results indicate that the mouse M-type receptor is selective for only a subset of mouse sPLA2s from the group I/II/V/X structural collection. Binding of mouse sPLA2s to a recombinant soluble mouse M-type receptor leads in all cases to inhibition of enzymatic activity, and the extent of deglycosylation of the receptor decreases yet does not abolish sPLA2 binding. The physiological meaning of binding of sPLA2 to the M-type receptor is discussed on the basis of our current knowledge of sPLA2 functions.

  10. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo.

    PubMed

    Erdmann, Andreas A; Gao, Zhan-Guo; Jung, Unsu; Foley, Jason; Borenstein, Todd; Jacobson, Kenneth A; Fowler, Daniel H

    2005-06-15

    To evaluate the direct effect of adenosine on cytokine-polarized effector T cells, murine type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1) and Th2/Tc2 cells were generated using an antigen-presenting cell (APC)-free method. Tc1 and Tc2 cells had similar adenosine signaling, as measured by intracellular cyclic AMP (cAMP) increase upon adenosine A(2A) receptor agonism by CGS21680 (CGS). CGS greatly reduced Tc1 and Tc2 cell interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) secretion, with nominal effect on interferon gamma (IFN-gamma) secretion. Tc2 cell IL-4 and IL-5 secretion was not reduced by CGS, and IL-10 secretion was moderately reduced. Agonist-mediated inhibition of IL-2 and TNF-alpha secretion occurred via A(2A) receptors, with no involvement of A(1), A(2B), or A(3) receptors. Adenosine agonist concentrations that abrogated cytokine secretion did not inhibit Tc1 or Tc2 cell cytolytic function. Adenosine modulated effector T cells in vivo, as CGS administration reduced CD4(+)Th1 and CD8(+)Tc1 cell expansion to alloantigen and, in a separate model, reduced antigen-specific CD4(+) Th1 cell numbers. Remarkably, agonist-mediated T-cell inhibition was abrogated by in vivo IL-2 therapy. Adenosine receptor activation therefore preferentially inhibits type I cytokine secretion, most notably IL-2. Modulation of adenosine receptors may thus represent a suitable target primarily for inflammatory conditions mediated by Th1 and Tc1 cells.

  11. Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

    PubMed

    Gilbert, J J; Stewart, A; Courtney, C A; Fleming, M C; Reid, P; Jackson, C G; Wise, A; Wakelam, M J; Harnett, M M

    1996-03-15

    The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

  12. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  13. Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors

    PubMed Central

    Viana da Silva, Silvia; Haberl, Matthias Georg; Zhang, Pei; Bethge, Philipp; Lemos, Cristina; Gonçalves, Nélio; Gorlewicz, Adam; Malezieux, Meryl; Gonçalves, Francisco Q.; Grosjean, Noëlle; Blanchet, Christophe; Frick, Andreas; Nägerl, U Valentin; Cunha, Rodrigo A.; Mulle, Christophe

    2016-01-01

    Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients. PMID:27312972

  14. Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, h...

  15. Synthesis and Preliminary Evaluation of a 2-Oxoquinoline Carboxylic Acid Derivative for PET Imaging the Cannabinoid Type 2 Receptor

    PubMed Central

    Mu, Linjing; Slavik, Roger; Müller, Adrienne; Popaj, Kasim; Čermak, Stjepko; Weber, Markus; Schibli, Roger; Krämer, Stefanie D.; Ametamey, Simon M.

    2014-01-01

    Cannabinoid receptor subtype 2 (CB2) has been shown to be up-regulated in activated microglia and therefore plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer’s disease. The CB2 receptor is therefore considered as a very promising target for therapeutic approaches as well as for imaging. A promising 2-oxoquinoline derivative designated KP23 was synthesized and radiolabeled and its potential as a ligand for PET imaging the CB2 receptor was evaluated. [11C]KP23 was obtained in 10%–25% radiochemical yield (decay corrected) and 99% radiochemical purity. It showed high stability in phosphate buffer, rat and mouse plasma. In vitro autoradiography of rat and mouse spleen slices, as spleen expresses a high physiological expression of CB2 receptors, demonstrated that [11C]KP23 exhibits specific binding towards CB2. High spleen uptake of [11C]KP23 was observed in dynamic in vivo PET studies with Wistar rats. In conclusion, [11C]KP23 showed promising in vitro and in vivo characteristics. Further evaluation with diseased animal model which has higher CB2 expression levels in the brain is warranted. PMID:24662272

  16. The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices

    PubMed Central

    Pugliese, AM; Traini, C; Cipriani, S; Gianfriddo, M; Mello, T; Giovannini, MG; Galli, A; Pedata, F

    2009-01-01

    Background and purpose: Activation of adenosine A2A receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated. Experimental approach: We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots. Key results: OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A2A receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100–500 nmol·L−1) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A2A receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmol·L−1 ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmol·L−1 ZM241385. Conclusions and implications: In the CA1 hippocampus, antagonism of A2A adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival. PMID:19422385

  17. Transcriptomic Analysis Shows Decreased Cortical Expression of NR4A1, NR4A2 and RXRB in Schizophrenia and Provides Evidence for Nuclear Receptor Dysregulation

    PubMed Central

    Corley, Susan M.; Wilkins, Marc R.; Shannon Weickert, Cynthia

    2016-01-01

    Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort (p<0.05 and p<0.01, respectively). We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes. PMID:27992436

  18. T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells.

    PubMed

    Hillerdal, Victoria; Nilsson, Berith; Carlsson, Björn; Eriksson, Fredrik; Essand, Magnus

    2012-09-25

    To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2-restricted T-cell receptor γ-chain alternate reading-frame protein (TARP)(4-13) epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2(+) prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP(4-13) epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.

  19. Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited β-adrenergic mucin secretion response

    PubMed Central

    Pereira, M M C; Lloyd Mills, C; Dormer, R L; McPherson, M A

    1998-01-01

    The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated β-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β-adrenergic response and whether excessive cyclic AMP rise is required.CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis. PMID:9831904

  20. Genetic deletion of the adenosine A(2A) receptor prevents nicotine-induced upregulation of α7, but not α4β2* nicotinic acetylcholine receptor binding in the brain.

    PubMed

    Metaxas, Athanasios; Al-Hasani, Ream; Farshim, Pamela; Tubby, Kristina; Berwick, Amy; Ledent, Catherine; Hourani, Susanna; Kitchen, Ian; Bailey, Alexis

    2013-08-01

    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.

  1. Role and Function of A2A and A₃ Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis.

    PubMed

    Ravani, Annalisa; Vincenzi, Fabrizio; Bortoluzzi, Alessandra; Padovan, Melissa; Pasquini, Silvia; Gessi, Stefania; Merighi, Stefania; Borea, Pier Andrea; Govoni, Marcello; Varani, Katia

    2017-03-24

    Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A₃AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A₃ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A₃AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A2A and A₃AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A₃ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

  2. Apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine for adenosine A1 and A2 receptors in isolated tissues from guinea-pigs.

    PubMed Central

    Collis, M. G.; Stoggall, S. M.; Martin, F. M.

    1989-01-01

    1. The classification of adenosine receptor subtypes (A1 and A2) in intact tissues has been based on the order of agonist potency. In this study the apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine (CPX), an antagonist which has been reported to be A1 selective, and the non-selective antagonist 1,3-dimethyl-8-phenylxanthine (8PT) has been evaluated on isolated tissues from the guinea-pig. 2. The isolated tissues used were atria (bradycardic response, proposed A1 sub-type), aorta and trachea (relaxant response, proposed A2 sub-type). 3. Both the xanthines antagonized responses to adenosine in the three tissues but had little or no effect on responses to carbachol (atria), sodium nitrite (aorta) or isoprenaline (trachea). 4. pA2 values for 8PT were similar on the three tissues (6.3-6.7), however, the pA2 value for CPX on the atria (7.9-8.4) was greater than that on the aorta (6.6) or trachea (6.6). 5. These results support the suggestion that the adenosine receptors which mediate bradycardia in the atrium are of the A1 sub-type and that those which mediate relaxation in the aorta and trachea are of the A2 type. PMID:2790383

  3. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Ng, Seng Kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2015-05-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR(+/-)) significantly protect ESMN from SOD1G93A(+) astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS.

  4. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Ng, Seng kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR+/−) significantly protect ESMN from SOD1G93A+ astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS. PMID:25779930

  5. The role of adenosine A1 and A2A receptors in the caffeine effect on MDMA-induced DA and 5-HT release in the mouse striatum.

    PubMed

    Górska, A M; Gołembiowska, K

    2015-04-01

    3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") popular as a designer drug is often used with caffeine to gain a stronger stimulant effect. MDMA induces 5-HT and DA release by interaction with monoamine transporters. Co-administration of caffeine and MDMA may aggravate MDMA-induced toxic effects on DA and 5-HT terminals. In the present study, we determined whether caffeine influences DA and 5-HT release induced by MDMA. We also tried to find out if adenosine A1 and A2A receptors play a role in the effect of caffeine by investigating the effect of the selective adenosine A1 and A2A receptor antagonists, DPCPX and KW 6002 on DA and 5-HT release induced by MDMA. Mice were treated with caffeine (10 mg/kg) and MDMA (20 or 40 mg/kg) alone or in combination. DA and 5-HT release in the mouse striatum was measured using in vivo microdialysis. Caffeine exacerbated the effect of MDMA on DA and 5-HT release. DPCPX or KW 6002 co-administered with MDMA had similar influence as caffeine, but KW 6002 was more potent than caffeine or DPCPX. To exclude the contribution of MAO inhibition by caffeine in the caffeine effect on MDMA-induced increase in DA and 5-HT, we also tested the effect of the nonxanthine adenosine receptor antagonist CGS 15943A lacking properties of MAO activity modification. Our findings indicate that adenosine A1 and A2A receptor blockade may account for the caffeine-induced exacerbation of the MDMA effect on DA and 5-HT release and may aggravate MDMA toxicity.

  6. Activation of the A2B adenosine receptor in B16 melanomas induces CXCL12 expression in FAP-positive tumor stromal cells, enhancing tumor progression

    PubMed Central

    Sorrentino, Claudia; Miele, Lucio; Porta, Amalia; Pinto, Aldo; Morello, Silvana

    2016-01-01

    The A2B receptor (A2BR) can mediate adenosine-induced tumor proliferation, immunosuppression and angiogenesis. Targeting the A2BR has proved to be therapeutically effective in some murine tumor models, but the mechanisms of these effects are still incompletely understood. Here, we report that pharmacologic inhibition of A2BR with PSB1115, which inhibits tumor growth, decreased the number of fibroblast activation protein (FAP)-expressing cells in tumors in a mouse model of melanoma. This effect was associated with reduced expression of fibroblast growth factor (FGF)-2. Treatment of melanoma-associated fibroblasts with the A2BR agonist Bay60-6583 enhanced CXCL12 and FGF2 expression. This effect was abrogated by PSB1115. The A2AR agonist CGS21680 did not induce CXCL12 or FGF2 expression in tumor associated fibroblasts. Similar results were obtained under hypoxic conditions in skin-derived fibroblasts, which responded to Bay60-6583 in an A2BR-dependent manner, by stimulating pERK1/2. FGF2 produced by Bay60-6583-treated fibroblasts directly enhanced the proliferation of melanoma cells. This effect could be reversed by PSB1115 or an anti-FGF2 antibody. Interestingly, melanoma growth in mice receiving Bay60-6583 was attenuated by inhibition of the CXCL12/CXCR4 pathway with AMD3100. CXCL12 and its receptor CXCR4 are involved in angiogenesis and immune-suppression. Treatment of mice with AMD3100 reduced the number of CD31+ cells induced by Bay60-6583. Conversely, CXCR4 blockade did not affect the accumulation of tumor-infiltrating MDSCs or Tregs. Together, our data reveal an important role for A2BR in stimulating FGF2 and CXCL12 expression in melanoma-associated fibroblasts. These factors contribute to create a tumor-promoting microenvironment. Our findings support the therapeutic potential of PSB1115 for melanoma. PMID:27590504

  7. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats

    PubMed Central

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  8. A(2A) adenosine receptors are differentially modulated by pharmacological treatments in rheumatoid arthritis patients and their stimulation ameliorates adjuvant-induced arthritis in rats.

    PubMed

    Vincenzi, Fabrizio; Padovan, Melissa; Targa, Martina; Corciulo, Carmen; Giacuzzo, Sarah; Merighi, Stefania; Gessi, Stefania; Govoni, Marcello; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    A(2A) adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A(2A)ARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A(2A)AR stimulation in a rat model of arthritis. We investigated A(2A)AR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A(2A)ARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A(2A)AR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A(2A)AR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A(2A)AR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A(2A)AR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A(2A)AR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A(2A)ARs in lymphocytes from RA patients. The effectiveness of A(2A)AR stimulation in a rat model of arthritis supported the role of A(2A)AR agonists as potential pharmacological treatment for RA.

  9. Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1.

    PubMed

    Köröskényi, Krisztina; Kiss, Beáta; Szondy, Zsuzsa

    2016-07-01

    Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

  10. Interleukin-1beta but not tumor necrosis factor-alpha potentiates neuronal damage by quinolinic acid: protection by an adenosine A2A receptor antagonist.

    PubMed

    Stone, Trevor W; Behan, Wilhelmina M H

    2007-04-01

    Quinolinic acid is an agonist at glutamate receptors sensitive to N-methyl-D-aspartate (NMDA). It has been implicated in neural dysfunction associated with infections, trauma, and ischemia, although its neurotoxic potency is relatively low. This study was designed to examine the effects of a combination of quinolinic acid and the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Compounds were administered to the hippocampus of anesthetized male rats, animals being allowed to recover for 7 days before histological analysis of the hippocampus for neuronal damage estimated by counting of intact, healthy neurons. A low dose of quinolinic acid or IL-1beta produced no damage by itself, but the two together induced a significant loss of pyramidal neurons in the hippocampus. Higher doses produced almost total loss of pyramidal cells. Intrahippocampal TNF-alpha produced no effect alone but significantly reduced the neuronal loss produced by quinolinic acid. The adenosine A(2A) receptor antagonist ZM241385 reduced neuronal loss produced by the combinations of quinolinic acid and IL-1beta. The results suggest that simultaneous quinolinic acid and IL-1beta, both being induced by cerebral infection or injury, are synergistic in the production of neuronal damage and could together contribute substantially to traumatic, infective, or ischemic cerebral damage. Antagonism of adenosine A(2A) receptors protects neurons against the combination of quinolinic acid and IL-1beta.

  11. Roles of the AMPA receptor subunit GluA1 but not GluA2 in synaptic potentiation and activation of ERK in the anterior cingulate cortex.

    PubMed

    Toyoda, Hiroki; Zhao, Ming-Gao; Ulzhöfer, Bettina; Wu, Long-Jun; Xu, Hui; Seeburg, Peter H; Sprengel, Rolf; Kuner, Rohini; Zhuo, Min

    2009-08-10

    Cortical areas including the anterior cingulate cortex (ACC) are important for pain and pleasure. Recent studies using genetic and physiological approaches have demonstrated that the investigation of basic mechanism for long-term potentiation (LTP) in the ACC may reveal key cellular and molecular mechanisms for chronic pain in the cortex. Glutamate N-methyl D-aspartate (NMDA) receptors in the ACC are critical for the induction of LTP, including both NR2A and NR2B subunits. However, cellular and molecular mechanisms for the expression of ACC LTP have been less investigated. Here, we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, GluA1 but not GluA2 contributes to LTP in the ACC using genetic manipulated mice lacking GluA1 or GluA2 gene. Furthermore, GluA1 knockout mice showed decreased extracellular signal-regulated kinase (ERK) phosphorylation in the ACC in inflammatory pain models in vivo. Our results demonstrate that AMPA receptor subunit GluA1 is a key mechanism for the expression of ACC LTP and inflammation-induced long-term plastic changes in the ACC.

  12. BDNF prevents NMDA-induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved.

    PubMed

    Martire, Alberto; Pepponi, Rita; Domenici, Maria Rosaria; Ferrante, Antonella; Chiodi, Valentina; Popoli, Patrizia

    2013-04-01

    NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.

  13. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    PubMed

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  14. A simple, quick, and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies.

    PubMed

    Ruan, Ke-He; Cervantes, Vanessa; Wu, Jiaxin

    2008-07-01

    Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full-sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full-sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by different detergents for comparison and was finally purified to a nearly single band with a ratio of 1:0.9 +/- 0.05 (ligand:receptor molecule) in ligand binding using a Ni column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNA digestion, dodecyl-maltoside detergent extraction, centrifugation, and FPLC purification. The purity and quantity of the purified TP, using the high-yield approach, were suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution one-dimensional and two-dimensional (1)H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.

  15. Opposite modulation of 4-aminopyridine and hypoxic hyperexcitability by A1 and A2 adenosine receptor ligands in rat hippocampal slices.

    PubMed

    Longo, R; Zeng, Y C; Sagratella, S

    1995-11-10

    The effects of the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), and of the adenosine agonists N6-cyclopentyladenosine (CPA), N6-(2-phenylisopropyl)adenosine (R-PIA), and 2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680) were investigated on the hyperexcitability induced in the CA1 area of rat hippocampal slices by hypoxia or the epileptogenic agent 4-aminopiridine. Slice perfusion with the mixed adenosine receptor agonist R-PIA (0.2 microM) significantly (P < 0.05) decreased: (i) the number of slices showing a transient CA1 epileptiform bursting during the hypoxic period; (ii) the duration of the hypoxia-induced epileptiform bursting. Conversely, slice perfusion with the selective A1 adenosine receptor antagonists DPCPX (0.2 microM) or with the selective A2 adenosine receptor agonist CGS 21680 significantly (P < 0.05) increased the number of slices showing a transient CA1 epileptiform bursting during the hypoxic period but did not affect the duration of the hypoxia-induced epileptiform bursting. Neither drug significantly affected the number of slices showing functional recovery after hypoxia. Slice perfusion with DPCPX (0.2 microM) also significantly increased (P < 0.05) the number of slices showing a persistent CA1 epileptiform bursting during the reoxygenation period, while the other drugs failed to affect it. Slice perfusion with the selective A1 adenosine receptor agonist CPA (2 microM) or R-PIA (5 microM) significantly (P < 0.05) decreased the duration of the CA1 epileptiform bursting induced by 100 microM 4-aminopyridine. CGS 21680 (5 microM) perfused together with CPA (2 microM) significantly (P < 0.05) counteracted the inhibitory effects of the A1 adenosine receptor agonist on 4-aminopyridine epileptiform bursting, while it failed by itself to directly affect the 4-aminopyridine epileptiform bursting duration. The results produce evidence for a selective opposite modulation by A1 and A2 adenosine

  16. Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors.

    PubMed

    López-Cruz, Laura; San-Miguel, Noemí; Bayarri, Pilar; Baqi, Younis; Müller, Christa E; Salamone, John D; Correa, Mercé

    2016-01-01

    Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0-1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0-60.0 mg/kg), and even blocked social preference at higher doses (30.0-60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3-9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5-6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5-1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0-30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol impairs the

  17. Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors

    PubMed Central

    López-Cruz, Laura; San-Miguel, Noemí; Bayarri, Pilar; Baqi, Younis; Müller, Christa E.; Salamone, John D.; Correa, Mercé

    2016-01-01

    Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0–1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0–60.0 mg/kg), and even blocked social preference at higher doses (30.0–60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3–9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5–6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5–1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0–30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol

  18. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  19. The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann-Pick type C patients.

    PubMed

    Visentin, Sergio; De Nuccio, Chiara; Bernardo, Antonietta; Pepponi, Rita; Ferrante, Antonella; Minghetti, Luisa; Popoli, Patrizia

    2013-09-25

    Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.

  20. Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2A and A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts

    PubMed Central

    Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Gessi, Stefania; Merighi, Stefania; Setti, Stefania; Cadossi, Ruggero; Goldring, Mary B.; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders

  1. Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride.

    PubMed

    Giunta, Salvatore; Andriolo, Violetta; Castorina, Alessandro

    2014-09-01

    In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl3) enhanced the neurotoxicity of the amyloid beta(25-35) fragment (Abeta(25-35)) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A2A adenosine receptors (A2AR), although it also inhibits A1Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta(25-35)+AlCl3) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta(25-35)+AlCl3 were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A1R and A2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl3 treatment exacerbated Abeta(25-35) toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta(25-35) only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta(25-35)+AlCl3-induced damage, suggesting that A1Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A1R inhibition in the setting of combined Abeta(25-35)+AlCl3 neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum.

  2. Pharmacokinetic modelling of the haemodynamic effects of the A2a adenosine receptor agonist CGS 21680C in conscious normotensive rats.

    PubMed Central

    Mathôt, R A; Cleton, A; Soudijn, W; IJzerman, A P; Danhof, M

    1995-01-01

    1. The aim of the present investigation was to determine the relationship between the blood concentration and haemodynamic effects of the adenosine A2a receptor agonist, CGS 21680C (the sodium salt of 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamidoadeno sin e) in conscious normotensive rats. 2. Chronically cannulated rats were randomly assigned to three groups which received 300, 1000 or 3000 micrograms kg-1 (0.56, 1.9 or 5.6 mumol kg-1) of CGS 21680C intravenously over 15 min. The mean arterial blood pressure (MAP) and heart rate (HR) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3. For each individual rat the reduction in mean arterial pressure was related to the blood concentration according to the sigmoidal Emax model. The concentration-effect relationships were consistent for the different treatment groups. The potency based on free drug concentrations (EC50,u) was 5.8 ng ml-1 (11 nM) (mean +/- s.e.; n = 19) and correlated well with the reported adenosine A2a receptor affinity (Ki 19 nM). In comparison with the reduction in blood pressure, CGS 21680C exhibited a greater potency for the reduction of the ratio MAP/HR. 4. It is concluded that estimates can be obtained for the potency and intrinsic activity of adenosine A2a receptor agonists in vivo by pharmacokinetic-pharmacodynamic analysis of mean arterial pressure data in a rat model.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7773536

  3. Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: role for the adenosine A(2A) receptor.

    PubMed

    Ribeiro, Alison; Ferraz-de-Paula, Viviane; Pinheiro, Milena L; Vitoretti, Luana B; Mariano-Souza, Domenica P; Quinteiro-Filho, Wanderley M; Akamine, Adriana T; Almeida, Vinícius I; Quevedo, João; Dal-Pizzol, Felipe; Hallak, Jaime E; Zuardi, Antônio W; Crippa, José A; Palermo-Neto, João

    2012-03-05

    Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor.

  4. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  5. Effect of subtype-selective adenosine receptor antagonists on basal or haloperidol-regulated striatal function: studies of exploratory locomotion and c-Fos immunoreactivity in outbred and A(2A)R KO mice.

    PubMed

    Pardo, M; López-Cruz, L; Valverde, O; Ledent, C; Baqi, Y; Müller, C E; Salamone, J D; Correa, M

    2013-06-15

    Behavioral activation is regulated by dopamine (DA) in striatal areas. At low doses, while typical antipsychotic drugs produce psychomotor slowing, psychostimulants promote exploration. Minor stimulants such as caffeine, which act as adenosine receptor antagonists, can also potentiate behavioral activation. Striatal areas are rich in adenosine and DA receptors, and adenosine A2A receptors are mainly expressed in the striatum where they are co-localized with DA D2 receptors. Adenosine antagonists with different receptor-selectivity profiles were used to study spontaneous or haloperidol-impaired exploration and c-Fos expression in different striatal areas. Because A2A antagonists were expected to be more selective for reversing the effects of the D2 antagonist haloperidol, A2A receptor knockout (A2ARKO) mice were also assessed. CD1 and A2ARKO male mice were tested in an open field and in a running wheel. Only the A1/A2A receptor antagonist theophylline (5.0-15.0 mg/kg) and the A2A antagonist MSX-3 (2.0 mg/kg) increased spontaneous locomotion and rearing. Co-administration of theophylline (10.0-15.0 mg/kg), and MSX-3 (1.0-3.0 mg/kg) reversed haloperidol-induced suppression of locomotion. The A1 antagonist CPT was only marginally effective in reversing the effects of haloperidol. Although adenosine antagonists did not affect c-Fos expression on their own, theophylline and MSX-3, but not CPT, attenuated haloperidol induction of c-Fos expression. A2ARKO mice were resistant to the behavioral effects of haloperidol at intermediate doses (0.1 mg/kg) in the open field and in the running wheel. A2A receptors are important for regulating behavioral activation, and interact with D2 receptors in striatal areas to regulate neural processes involved in exploratory activity.

  6. The effect of caffeine to increase reaction time in the rat during a test of attention is mediated through antagonism of adenosine A2A receptors.

    PubMed

    Higgins, Guy A; Grzelak, Michael E; Pond, Annamarie J; Cohen-Williams, Mary E; Hodgson, Robert A; Varty, Geoffrey B

    2007-12-11

    Caffeine produces effects on cognitive function particularly relating to aspects of attention such as reaction time. Considering the plasma exposure levels following regular caffeine intake, and the affinity of caffeine for known protein targets, these effects are likely mediated by either the adenosine A(1) or A(2A) receptor. In the present studies, two rat strains [Long-Evans (LE) and CD] were trained to asymptote performance in a test of selective attention, the 5-choice serial reaction time task (5-CSRTT). Next, the effects of caffeine were compared to the selective A(2A) antagonists, SCH 412348 and KW-6002 (Istradefylline), and the A(1) antagonist, DPCPX. Further studies compared the psychostimulant effects of each drug. Finally, we tested the A(2A) agonist, CGS-21680, on 5-CSRTT performance and given the antipsychotic potential of this drug class, studied the interaction between CGS-21680 and amphetamine in this task. Caffeine (3-10mg/kg IP) increased reaction time in both LE and CD rats, with no effect on accuracy, an effect replicated by SCH 412348 (0.1-1mg/kg PO) and KW-6002 (1-3mg/kg PO), but not DPCPX (3-30 mg/kg PO). At least with SCH 412348, these effects were at doses that were not overtly psychostimulant. In contrast, CGS-21680 (0.03-0. 3mg/kg IP) slowed reaction speed and increased omissions. Interestingly, at a comparatively low dose of 0.03 mg/kg, CGS-21680 attenuated the increased premature responding produced by amphetamine (1mg/kg IP). The present results suggest that the attention-enhancing effects of caffeine are mediated through A(2A) receptor blockade, and selective A(2A) receptor antagonists may have potential as therapies for attention-related disorders. Furthermore, the improvement in response control in amphetamine-treated rats following CGS-21680 pretreatment supports the view that A(2A) agonists have potential as novel antipsychotics.

  7. Cocaine-Induced Changes of Synaptic Transmission in the Striatum are Modulated by Adenosine A2A Receptors and Involve the Tyrosine Phosphatase STEP

    PubMed Central

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria

    2014-01-01

    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine. PMID:23989619

  8. Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

    PubMed

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria

    2014-02-01

    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

  9. Association of rare variation in the glutamate receptor gene SLC1A2 with susceptibility to bipolar disorder and schizophrenia.

    PubMed

    Fiorentino, Alessia; Sharp, Sally I; McQuillin, Andrew

    2015-09-01

    The SLC1A2 gene encodes the excitatory amino acid transporter 2 (EAAT2). Glutamate is the major mediator of excitatory neurotransmission and EAAT2 is responsible for clearing the neurotransmitter from the synaptic cleft. Genetic variation in SLC1A2 has been implicated in a range of neurological and neuropsychiatric conditions including schizophrenia (SZ), autism and in core phenotypes of bipolar disorder (BD). The coding and putative regulatory regions of SLC1A2 gene were screened for variants using high resolution melting or sequenced in 1099 or in 32 BD subjects. Thirty-two variants were detected in the SLC1A2 gene. Fifteen potentially etiological variants were selected for genotyping in 1099 BD and 1095 control samples. Five amino acid changing variants were also genotyped in 630 participants suffering from SZ. None of the variants were found to be associated with BD or SZ or with the two diseases combined. However, two recurrent missense variants (rs145827578:G>A, p.(G6S); rs199599866:G>A, p.(R31Q)) and one recurrent 5'-untranslated region (UTR) variant (ss825678885:G>T) were detected in cases only. Combined analysis of the recurrent-case-only missense variants and of the case-only missense and 5'-UTR variants showed nominal evidence for association with the combined diseases (Fisher's P=0.019 and 0.0076). These findings are exploratory in nature and await replication in larger cohorts, however, they provide intriguing evidence that potentially functional rare variants in the SLC1A2 gene may confer susceptibility to psychotic disorders.

  10. Spatiotemporal brain dynamics of emotional face processing modulations induced by the serotonin 1A/2A receptor agonist psilocybin.

    PubMed

    Bernasconi, Fosco; Schmidt, André; Pokorny, Thomas; Kometer, Michael; Seifritz, Erich; Vollenweider, Franz X

    2014-12-01

    Emotional face processing is critically modulated by the serotonergic system. For instance, emotional face processing is impaired by acute psilocybin administration, a serotonin (5-HT) 1A and 2A receptor agonist. However, the spatiotemporal brain mechanisms underlying these modulations are poorly understood. Here, we investigated the spatiotemporal brain dynamics underlying psilocybin-induced modulations during emotional face processing. Electrical neuroimaging analyses were applied to visual evoked potentials in response to emotional faces, following psilocybin and placebo administration. Our results indicate a first time period of strength (i.e., Global Field Power) modulation over the 168-189 ms poststimulus interval, induced by psilocybin. A second time period of strength modulation was identified over the 211-242 ms poststimulus interval. Source estimations over these 2 time periods further revealed decreased activity in response to both neutral and fearful faces within limbic areas, including amygdala and parahippocampal gyrus, and the right temporal cortex over the 168-189 ms interval, and reduced activity in response to happy faces within limbic and right temporo-occipital brain areas over the 211-242 ms interval. Our results indicate a selective and temporally dissociable effect of psilocybin on the neuronal correlates of emotional face processing, consistent with a modulation of the top-down control.

  11. Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution

    PubMed Central

    Mamrosh, Jennifer L; Lee, Jae Man; Wagner, Martin; Stambrook, Peter J; Whitby, Richard J; Sifers, Richard N; Wu, San-Pin; Tsai, Ming-Jer; DeMayo, Francesco J; Moore, David D

    2014-01-01

    Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress. DOI: http://dx.doi.org/10.7554/eLife.01694.001 PMID:24737860

  12. Adenosine A2 receptor activation ameliorates mitochondrial oxidative stress upon reperfusion through the posttranslational modification of NDUFV2 subunit of complex I in the heart.

    PubMed

    Xu, Jingman; Bian, Xiyun; Liu, Yuan; Hong, Lan; Teng, Tianming; Sun, Yuemin; Xu, Zhelong

    2017-05-01

    While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I

  13. Opposite effects of the A2A receptor agonist CGS21680 in the striatum of Huntington's disease versus wild-type mice.

    PubMed

    Martire, Alberto; Calamandrei, Gemma; Felici, Fabio; Scattoni, Maria Luisa; Lastoria, Giusi; Domenici, Maria Rosaria; Tebano, Maria Teresa; Popoli, Patrizia

    2007-04-24

    Huntington's disease (HD) is an inherited neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) are involved in excitotoxic/neurodegenerative processes, and A(2A)R ligands may be neuroprotective in models of HD. However, changes in the transcription, expression and function of A(2A)Rs have been reported to occur in HD models. The aim of the present work was to verify whether A(2A)R-mediated effects are altered in the striatum of transgenic HD (R6/2) versus wild-type (WT) mice. Extracellular field potentials (FPs) were recorded in corticostriatal slices from R6/2 mice in early (7-8 weeks) or frankly (12-13 weeks) symptomatic phases, and age-matched WT. In 12-13 weeks aged WT animals, the application of 75 microM NMDA induced a transient disappearance of the FP followed by an almost complete recovery at washout. In slices from HD mice, the mean FP recovery was significantly reduced (P<0.01 versus WT). A(2A)R activation oppositely modulated NMDA-induced toxicity in the striatum of HD versus WT mice. Indeed, the A(2A)R agonist CGS21680 reduced the FP recovery in slices from WT mice, while it significantly increased it in slices from R6/2 mice. In early symptomatic (7-8 weeks) mice, no differences were observed between WT and HD animals in terms of basal synaptic transmission and response to NMDA. At the same age, the behavioural effects elicited by CGS21680 were qualitatively identical in WT and HD mice. These findings may have very important implications for the neuroprotective potential of A(2A)R ligands in HD.

  14. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 1. Structure-activity relationships and optimization of heterocyclic substituents.

    PubMed

    Slee, Deborah H; Chen, Yongsheng; Zhang, Xiaohu; Moorjani, Manisha; Lanier, Marion C; Lin, Emily; Rueter, Jaimie K; Williams, John P; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Santos, Mark; Gross, Raymond S; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Saunders, John

    2008-03-27

    Previously we have described a novel series of potent and selective A 2A receptor antagonists (e.g., 1) with excellent aqueous solubility. While these compounds are efficacious A 2A antagonists in vivo, the presence of an unsubstituted furyl moiety was a cause of some concern. In order to avoid the potential metabolic liabilities that could arise from an unsubstituted furyl moiety, an optimization effort was undertaken with the aim of replacing the unsubstituted furan with a more metabolically stable group while maintaining potency and selectivity. Herein, we describe the synthesis and SAR of a range of novel heterocyclic systems and the successful identification of a replacement for the unsubstituted furan moiety with a methylfuran or thiazole moiety while maintaining potency and selectivity.

  15. GluA2-Containing AMPA Receptors Distinguish Ribbon-Associated from Ribbonless Afferent Contacts on Rat Cochlear Hair Cells123

    PubMed Central

    Martinez-Monedero, Rodrigo

    2016-01-01

    Abstract Mechanosensory hair cells release glutamate at ribbon synapses to excite postsynaptic afferent neurons, via AMPA-type ionotropic glutamate receptors (AMPARs). However, type II afferent neurons contacting outer hair cells in the mammalian cochlea were thought to differ in this respect, failing to show GluA immunolabeling and with many “ribbonless” afferent contacts. Here it is shown that antibodies to the AMPAR subunit GluA2 labeled afferent contacts below inner and outer hair cells in the rat cochlea, and that synaptic currents in type II afferents had AMPAR-specific pharmacology. Only half the postsynaptic densities of type II afferents that labeled for PSD-95, Shank, or Homer were associated with GluA2 immunopuncta or presynaptic ribbons, the “empty slots” corresponding to ribbonless contacts described previously. These results extend the universality of AMPAergic transmission by hair cells, and support the existence of silent afferent contacts. PMID:27257620

  16. The adenosine A2A receptor agonist CGS 21680 decreases ethanol self-administration in both non-dependent and dependent animals.

    PubMed

    Houchi, Hakim; Persyn, Wolfgang; Legastelois, Rémi; Naassila, Mickaël

    2013-09-01

    There is emerging evidence that the adenosinergic system might be involved in drug addiction and alcohol dependence. We have already demonstrated the involvement of A2A receptors (A2AR) in ethanol-related behaviours in mice. Here, we investigated whether the A2AR agonist CGS 21680 can reduce ethanol operant self-administration in both non-dependent and ethanol-dependent Wistar rats. To rule out a potential involvement of the A1R in the effects of CGS 21680, we also tested its effectiveness to reduce ethanol operant self-administration in both heterozygous and homozygous A1R knockout mice. Our results demonstrated that CGS 21680 (0.065, 0.095 and 0.125 mg/kg, i.p.) had a bimodal effect on 10% ethanol operant self-administration in non-dependent rats. The intermediate dose was also effective in reducing 2% sucrose self-administration. Interestingly, the intermediate dose reduced 10% ethanol self-administration in dependent animals more effectively (75% decrease) when compared with non-dependent animals (57% decrease). These results suggest that the A2AR are involved in CGS 21680 effects since the reduction of ethanol self-administration was not dependent upon the presence of A1R in mice. In conclusion, our findings demonstrated the effectiveness of the A2AR agonist CGS 21680 in a preclinical model of alcohol addiction and suggested that the adenosinergic pathway is a promising target to treat alcohol addiction.

  17. Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

    PubMed Central

    Keasey, M. P.; Lemos, R. R.; Hagg, T.; Oliveira, J. R. M.

    2016-01-01

    Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications (“Fahr’s disease”). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr’s disease and normal aging. PMID:27184385

  18. Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.

    PubMed

    Goodwill, Adam G; James, Milinda E; Frisbee, Jefferson C

    2008-10-01

    This study determined if altered vascular prostacyclin (PGI(2)) and/or thromboxane A(2) (TxA(2)) production with reduced Po(2) contributes to impaired hypoxic dilation of skeletal muscle resistance arterioles of obese Zucker rats (OZRs) versus lean Zucker rats (LZRs). Mechanical responses were assessed in isolated gracilis muscle arterioles following reductions in Po(2) under control conditions and following pharmacological interventions inhibiting arachidonic acid metabolism and nitric oxide synthase and alleviating elevated vascular oxidant stress. The production of arachidonic acid metabolites was assessed using pooled arteries from OZRs and LZRs in response to reduced Po(2). Hypoxic dilation, endothelium-dependent in both strains, was attenuated in OZRs versus LZRs. Nitric oxide synthase inhibition had no significant impact on hypoxic dilation in either strain. Cyclooxygenase inhibition dramatically reduced hypoxic dilation in LZRs and abolished responses in OZRs. Treatment of arterioles from OZRs with polyethylene glycol-superoxide dismutase improved hypoxic dilation, and this improvement was entirely cyclooxygenase dependent. Vascular PGI(2) production with reduced Po(2) was similar between strains, although TxA(2) production was increased in OZRs, a difference that was attenuated by treatment of vessels from OZRs with polyethylene glycol-superoxide dismutase. Both blockade of PGH(2)/TxA(2) receptors and inhibition of thromboxane synthase increased hypoxic dilation in OZR arterioles. These results suggest that a contributing mechanism underlying impaired hypoxic dilation of skeletal muscle arterioles of OZRs may be an increased vascular production of TxA(2), which competes against the vasodilator influences of PGI(2). These results also suggest that the elevated vascular oxidant stress inherent in metabolic syndrome may contribute to the increased vascular TxA(2) production and may blunt vascular sensitivity to PGI(2).

  19. Oral administration of Japanese sake yeast (Saccharomyces cerevisiae sake) promotes non-rapid eye movement sleep in mice via adenosine A2A receptors.

    PubMed

    Nakamura, Yoshitaka; Midorikawa, Tatsuyuki; Monoi, Noriyuki; Kimura, Eriko; Murata-Matsuno, Ayumi; Sano, Tomomi; Oka, Kengo; Sugafuji, Toshihiro; Uchiyama, Akira; Murakoshi, Michiaki; Sugiyama, Keikichi; Nishino, Hoyoku; Urade, Yoshihiro

    2016-12-01

    We have demonstrated previously that Japanese sake yeast improves sleep quality in humans. In the present study, we examined the molecular mechanisms of sake yeast to induce sleep by monitoring locomotor activity, electromyogram and electroencephalogram in mice. Oral administration of Japanese sake yeast (100, 200, and 300 mg kg(-1) ) decreased the locomotor activity by 18, 46 and 59% and increased the amount of non-rapid eye movement (NREM) sleep by 1.5-, 2.3- and 2.4-fold (to 37 ± 6, 57 ± 8, and 60 ± 4 min from 25 ± 6 min in the vehicle-administered group, respectively) in a dose-dependent manner for 4 h after oral administration. However, Japanese sake yeast did not change the amount of rapid eye movement (REM) sleep, the electroencephalogram power density during NREM sleep or show any adverse effects, such as rebound of insomnia, during 24 h postadministration and on the next day. An intraperitoneal pretreatment with an adenosine A2A receptor-selective antagonist, ZM241385 (15 mg kg(-1) ), reduced the amount of NREM sleep of sake yeast-administered mice to the basal level, without changing basal amount of sleep. Conversely, an A1 receptor-selective antagonist, 8-cyclopentyltheophylline (10 mg kg(-1) ), did not affect the sleep-promoting effect of Japanese sake yeast. Thus, Japanese sake yeast promotes NREM sleep via activation of adenosine A2A but not A1 receptors.

  20. A 5-HT2A/2C receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, mitigates developmental neurotoxicity of ethanol to serotonergic neurons.

    PubMed

    Ishiguro, Tsukasa; Sakata-Haga, Hiromi; Fukui, Yoshihiro

    2016-07-01

    Prenatal ethanol exposure causes the reduction of serotonergic (5-HTergic) neurons in the midbrain raphe nuclei. In the present study, we examined whether an activation of signaling via 5-HT2A and 5-HT2C receptors during the fetal period is able to prevent the reduction of 5-HTergic neurons induced by prenatal ethanol exposure. Pregnant Sprague-Dawley rats were given a liquid diet containing 2.5 to 5.0% (w/v) ethanol on gestational days (GDs) 10 to 20 (Et). As a pair-fed control, other pregnant rats were fed the same liquid diet except that the ethanol was replaced by isocaloric sucrose (Pf). Each Et and Pf group was subdivided into two groups; one of the groups was treated with 1 mg/kg (i.p.) of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), an agonist for 5-HT2A/2C receptors, during GDs 13 to 19 (Et-DOI or Pf-DOI), and another was injected with saline vehicle only (Et-Sal or Pf-Sal). Their fetuses were removed by cesarean section on GD 19 or 20, and fetal brains were collected. An immunohistological examination of 5-HTergic neurons in the fetuses on embryonic day 20 using an antibody against tryptophan hydroxylase revealed that the number of 5-HTergic neurons in the midbrain raphe nuclei was significantly reduced in the Et-Sal fetuses compared to that of the Pf-Sal and Pf-DOI fetuses, whereas there were no significant differences between Et-DOI and each Pf control. Thus, we concluded that the reduction of 5-HTergic neurons that resulted in prenatal ethanol exposure could be alleviated by the enhancement of signaling via 5-HT2A/2C receptors during the fetal period.

  1. Activity of 8F4, a T-cell receptor-like anti-PR1/HLA-A2 antibody, against primary human AML in vivo.

    PubMed

    Sergeeva, A; He, H; Ruisaard, K; St John, L; Alatrash, G; Clise-Dwyer, K; Li, D; Patenia, R; Hong, R; Sukhumalchandra, P; You, M J; Gagea, M; Ma, Q; Molldrem, J J

    2016-07-01

    The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high-affinity T-cell receptor-like murine monoclonal antibody, 8F4, that binds to the PR1/HLA-A2 complex, mediates lysis of AML and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting the possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML.

  2. The antidepressant-like effect of inosine in the FST is associated with both adenosine A1 and A 2A receptors.

    PubMed

    Kaster, Manuella P; Budni, Josiane; Gazal, Marta; Cunha, Mauricio P; Santos, Adair R S; Rodrigues, Ana Lúcia S

    2013-09-01

    Inosine is an endogenous purine nucleoside, which is formed during the breakdown of adenosine. The adenosinergic system was already described as capable of modulating mood in preclinical models; we now explored the effects of inosine in two predictive models of depression: the forced swim test (FST) and tail suspension test (TST). Mice treated with inosine displayed higher anti-immobility in the FST (5 and 50 mg/kg, intraperitoneal route (i.p.)) and in the TST (1 and 10 mg/kg, i.p.) when compared to vehicle-treated groups. These antidepressant-like effects started 30 min and lasted for 2 h after intraperitoneal administration of inosine and were not accompanied by any changes in the ambulatory activity in the open-field test. Both adenosine A1 and A2A receptor antagonists prevented the antidepressant-like effect of inosine in the FST. In addition, the administration of an adenosine deaminase inhibitor (1 and 10 mg/kg, i.p.) also caused an antidepressant-like effect in the FST. These results indicate that inosine possesses an antidepressant-like effect in the FST and TST probably through the activation of adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  3. Tetanic depression is overcome by tonic adenosine A2A receptor facilitation of L-type Ca2+ influx into rat motor nerve terminals

    PubMed Central

    Oliveira, Laura; Timóteo, M Alexandrina; Correia-de-Sá, Paulo

    2004-01-01

    Motor nerve terminals possess multiple voltage-sensitive calcium channels operating acetylcholine (ACh) release. In this study, we investigated whether facilitation of neuromuscular transmission by adenosine generated during neuronal firing was operated by Ca2+ influx via ‘prevalent’ P-type or via the recruitment of ‘silent’ L-type channels. The release of [3H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5 Hz (83 ± 4 × 103 disintegrations per minute per gram (d.p.m. g−1); n = 11) to 50 Hz (30 ± 3 × 103 d.p.m. g−1; n = 5). The P-type Ca2+ channel blocker, ω-agatoxin IVA (100 nm) reduced (by 40 ± 10%; n = 6) the release of [3H]ACh evoked by 50-Hz trains, while nifedipine (1 μm, an L-type blocker) was inactive. Tetanic depression was overcome (88 ± 6 × 103 d.p.m. g−1; n = 12) by stimulating the phrenic nerve with 50-Hz bursts (five bursts of 150 pulses, 20 s interburst interval). In these conditions, ω-agatoxin IVA (100 nm) failed to affect transmitter release, but nifedipine (1 μm) decreased [3H]ACh release by 21 ± 7% (n = 4). Inactivation of endogenous adenosine with adenosine deaminase (ADA, 0.5 U ml−1) reduced (by 54 ± 8%, n = 5) the release of [3H]ACh evoked with 50-Hz bursts. This effect was opposite to the excitatory actions of adenosine (0.5 mm), S-(p-nitrobenzyl)-6-thioinosine (5 μm, an adenosine uptake blocker) and CGS 21680C (3 nm, a selective A2A receptor agonist); as the A1 receptor agonist R-N6-phenylisopropyl adenosine (R-PIA, 300 nm) failed to affect the release of [3H]ACh, the results indicate that adenosine generated during 50-Hz bursts exerts an A2A-receptor-mediated tonus. The effects of ADA (0.5 U ml−1) and CGS 21680C (3 nm) were prevented by nifedipine (1 μm). Blocking tonic A2A receptor activation, with ADA (0.5 U ml−1) or 3,7-dimethyl-1-propargyl xanthine (10 μm, an A2A antagonist), recovered ω-agatoxin IVA (100 nm) inhibition and

  4. Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

    PubMed

    Machado, Nuno J; Simões, Ana Patrícia; Silva, Henrique B; Ardais, Ana Paula; Kaster, Manuella P; Garção, Pedro; Rodrigues, Diana I; Pochmann, Daniela; Santos, Ana Isabel; Araújo, Inês M; Porciúncula, Lisiane O; Tomé, Ângelo R; Köfalvi, Attila; Vaugeois, Jean-Marie; Agostinho, Paula; El Yacoubi, Malika; Cunha, Rodrigo A; Gomes, Catarina A

    2017-03-01

    Caffeine prophylactically prevents mood and memory impairments through adenosine A2A receptor (A2AR) antagonism. A2AR antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A2AR density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A2AR agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A2AR antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A2AR controlling synaptic glutamatergic function.

  5. Stimulation of adenosine A2A receptors reduces intracellular cholesterol accumulation and rescues mitochondrial abnormalities in human neural cell models of Niemann-Pick C1.

    PubMed

    Ferrante, A; De Nuccio, C; Pepponi, R; Visentin, S; Martire, A; Bernardo, A; Minghetti, L; Popoli, P

    2016-04-01

    Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 μM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.

  6. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    PubMed

    Takeuchi, Yusuke; Morise, Jyoji; Morita, Ippei; Takematsu, Hiromu; Oka, Shogo

    2015-01-01

    The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  7. Potent, Metabolically Stable 2-Alkyl-8-(2H-1,2,3-triazol-2-yl)-9H-adenines as Adenosine A2A Receptor Ligands.

    PubMed

    Pace, Silvia; Brogin, Giandomenico; Stasi, Maria Antonietta; Riccioni, Teresa; Borsini, Franco; Capocasa, Francesca; Manera, Francesco; Tallarico, Carlo; Grossi, Pietro; Vacondio, Federica; Bassi, Michele; Bartoccini, Francesca; Lucarini, Simone; Piersanti, Giovanni; Tarzia, Giorgio; Cabri, Walter; Minetti, Patrizia

    2015-07-01

    Inhibition of adenosine A2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson's disease (PD). We previously identified the triazolo-9H-purine, ST1535, as a potent A(2A)R antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω-1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω-1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A(2A)R was determined. Two compounds, (2-(3,3-dimethylbutyl)-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-amine (3 b) and 4-(6-amino-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-2-yl)-2-methylbutan-2-ol (3 c), exhibited good affinity against A(2A)R (Ki =0.4 nM and 2 nM, respectively) and high in vitro metabolic stability (89.5% and 95.3% recovery, respectively, after incubation with HLM for two hours).

  8. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  9. High coffee intake, but not caffeine, is associated with reduced estrogen receptor negative and postmenopausal breast cancer risk with no effect modification by CYP1A2 genotype.

    PubMed

    Lowcock, Elizabeth C; Cotterchio, Michelle; Anderson, Laura N; Boucher, Beatrice A; El-Sohemy, Ahmed

    2013-01-01

    Associations between caffeine and coffee consumption and breast cancer risk are uncertain, with studies suggesting inverse and null associations. Variation in cytochrome P450 1A2 (CYP1A2), a gene responsible for caffeine metabolism, may modify these associations. Cases (n = 3,062) were recruited through the Ontario Cancer Registry and controls (n = 3,427) through random digit dialing. Logistic regression was used to evaluate associations between breast cancer risk and intakes of 7 caffeine-containing items and total caffeine, and examine whether a genetic variant in CYP1A2 (rs762551) modified these associations. Analyses were stratified by estrogen receptor (ER), menopausal, and smoking status. Generally, coffee and caffeine were not associated with breast cancer risk; however, a significant reduction in risk was observed with the highest category of coffee consumption [≥5 cups per day vs. never, multivariate-adjusted odds ratio (MVOR) = 0.71, 95% confidence interval (CI): 0.51, 0.98]. Variant rs762551 did not modify associations. In stratified analyses, high coffee intake was associated with reduced risk of ER- (MVOR = 0.41, 95% CI: 0.19, 0.92) and postmenopausal breast cancer (MVOR = 0.63, 95% CI: 0.43, 0.94). High coffee consumption, but not total caffeine, may be associated with reduced risk of ER- and postmenopausal breast cancers, independent of CYP1A2 genotype. Further studies are needed to replicate these findings.

  10. L-DOPA disrupts adenosine A(2A)-cannabinoid CB(1)-dopamine D(2) receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: biochemical and behavioral studies.

    PubMed

    Pinna, Annalisa; Bonaventura, Jordi; Farré, Daniel; Sánchez, Marta; Simola, Nicola; Mallol, Josefa; Lluís, Carme; Costa, Giulia; Baqi, Younis; Müller, Christa E; Cortés, Antoni; McCormick, Peter; Canela, Enric I; Martínez-Pinilla, Eva; Lanciego, José L; Casadó, Vicent; Armentero, Marie-Therese; Franco, Rafael

    2014-03-01

    Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA.

  11. Remifentanil-induced preconditioning has cross-talk with A1 and A2B adenosine receptors in ischemic-reperfused rat heart.

    PubMed

    Lee, Yong-Cheol; Jung, Jiyoon; Park, Sang-Jin

    2016-01-01

    The purpose of this study was to determine whether there is a cross-talk between opioid receptors (OPRs) and adenosine receptors (ADRs) in remifentanil preconditioning (R-Pre) and, if so, to investigate the types of ADRs involved in the cross-talk. Isolated rat hearts received 30 min of regional ischemia followed by 2 hr of reperfusion. OPR and ADR antagonists were perfused from 10 min before R-Pre until the end of R-Pre. The heart rate, left ventricular developed pressure (LVDP),velocity of contraction (+dP/dtmax), and coronary flow (CF) were recorded. The area at risk and area of necrosis were measured. After reperfusion, the LVDP, +dP/dtmax,and CF showed a significant increase in the R-Pre group compared with the control group (no intervention before or after regional ischemia). These increases in the R-Pre group were blocked by naloxone, a nonspecific ADR antagonist, an A1 ADR antagonist, and an A2B ADR antagonist. The infarct size was reduced significantly in the R-Pre group compared with the control group. The infarct-reducing effect in the R-Pre group was blocked by naloxone, the nonspecific ADR antagonist, the A1 ADR antagonist, and the A2B ADR antagonist. The results of this study demonstrate that there is cross-talk between ADRs and OPRs in R-Pre and that A1 ADR and A2B ADR appear to be involved in the cross-talk.

  12. Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

    PubMed

    Campo, Giuseppe M; Micali, Antonio; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Pisani, Antonina; Bruschetta, Antongiulio; Calatroni, Alberto; Puzzolo, Domenico; Campo, Salvatore

    2015-05-01

    Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

  13. Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease

    PubMed Central

    Wallace, Kori L.

    2010-01-01

    We showed previously that pulmonary function and arterial oxygen saturation in NY1DD mice with sickle cell disease (SCD) are improved by depletion of invariant natural killer T (iNKT) cells or blockade of their activation. Here we demonstrate that SCD causes a 9- and 6-fold induction of adenosine A2A receptor (A2AR) mRNA in mouse pulmonary iNKT and natural killer (NK) cells, respectively. Treating SCD mice with the A2AR agonist ATL146e produced a dose-dependent reversal of pulmonary dysfunction with maximal efficacy at 10 ng/kg/minute that peaked within 3 days and persisted throughout 7 days of continuous infusion. Crossing NY1DD mice with Rag1−/− mice reduced pulmonary injury that was restored by adoptive transfer of 106 purified iNKT cells. Reconstituted injury was reversed by ATL146e unless the adoptively transferred iNKT cells were pretreated with the A2AR alkylating antagonist, FSPTP (5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ϵ]-1,2,4-triazolo[1,5-c]pyrimidine), which completely prevented pro-tection. In NY1DD mice exposed to hypoxia-reoxygenation, treatment with ATL146e at the start of reoxygenation prevented further lung injury. Together, these data indicate that activation of induced A2ARs on iNKT and NK cells in SCD mice is sufficient to improve baseline pulmonary function and prevent hypoxia-reoxygenation–induced exacerbation of pulmonary injury. A2A agonists have promise for treating diseases associated with iNKT or NK cell activation. PMID:20798237

  14. Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells.

    PubMed

    Shetty, Praveenkumar; Bargale, Anil; Patil, Basavraj R; Mohan, Rajashekar; Dinesh, U S; Vishwanatha, Jamboor K; Gai, Pramod B; Patil, Vidya S; Amsavardani, T S

    2016-01-01

    Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30 kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

  15. Involvement of cAMP-PKA pathway in adenosine A1 and A2A receptor-mediated regulation of acetaldehyde-induced activation of HSCs.

    PubMed

    Yang, Yaru; Wang, He; Lv, Xiongwen; Wang, Qi; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun

    2015-08-01

    The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

  16. Cigarette Smoke Impairs A2A Adenosine Receptor Mediated Wound Repair through Up-regulation of Duox-1 Expression

    PubMed Central

    Tian, Zhi; Zhang, Hui; Dixon, Jendayi; Traphagen, Nicole; Wyatt, Todd A.; Kharbanda, Kusum; Simet Chadwick, Samantha; Kolliputi, Narasaiah; Allen-Gipson, Diane S.

    2017-01-01

    Cigarette smoke (CS) exposure and intrinsic factors such as the NADPH oxidases produce high levels of reactive oxygen species (ROS), ensuing inflammatory tissue injury. We previously demonstrated that CS-generated ROS, particularly hydrogen peroxide (H2O2), impaired adenosine stimulated wound repair. We hypothesized that CS exposure modulates expression of Dual oxidase 1 (Duox-1), a NADPH oxidases known to generate H2O2. To test this hypothesis, we used human bronchial epithelial cell line Nuli-1 and C57BL/6 mice. Cells were treated with 5% CS extract (CSE) for various periods of time, and mice were exposed to whole body CS for six weeks. Both CSE and CS treatment induced increased expression of Duox-1, and silencing of Doux-1 improved the rate of cell wound repair induced by CSE treatment. Nuli-1 cells pretreated with thapsigargin but not calcium ionophore exhibited increased Duox-1 mRNA expression. CSE treatment stimulated PKCα activation, which was effectively blocked by pretreatment with diphenylene iodonium, a NADPH oxidase inhibitor. Compared to control, lungs from CS-exposed mice showed a significant increase in PKCα activity and Duox-1 expression. Collectively, the data demonstrated that CS exposure upregulates expression of Duox-1 protein. This further leads to H2O2 production and PKCα activation, inhibiting A2AAR-stimulated wound repair. PMID:28337995

  17. The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna

    2012-08-01

    It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson's disease (PD). In our previous study, we demonstrated that adenosine A(2A) receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A(2A) receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. L-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A(2A) receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage

  18. Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2)

    PubMed Central

    de Jesus Cortez, Felipe; Suzawa, Miyuki; Irvy, Sam; Bruning, John M.; Sablin, Elena; Jacobson, Matthew P.; Fletterick, Robert J.; Ingraham, Holly A.

    2016-01-01

    Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo. PMID:27467220

  19. Hypercalcemia stimulates expression of intrarenal phospholipase A2 and prostaglandin H synthase-2 in rats. Role of angiotensin II AT1 receptors.

    PubMed Central

    Mangat, H; Peterson, L N; Burns, K D

    1997-01-01

    In chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation. PMID:9329957

  20. Polydeoxyribonucleotide, an Adenosine-A2A Receptor Agonist, Preserves Blood Testis Barrier from Cadmium-Induced Injury

    PubMed Central

    Squadrito, Francesco; Micali, Antonio; Rinaldi, Mariagrazia; Irrera, Natasha; Marini, Herbert; Puzzolo, Domenico; Pisani, Antonina; Lorenzini, Cesare; Valenti, Andrea; Laurà, Rosaria; Germanà, Antonino; Bitto, Alessandra; Pizzino, Gabriele; Pallio, Giovanni; Altavilla, Domenica; Minutoli, Letteria

    2017-01-01

    Cadmium (Cd) impairs blood-testis barrier (BTB). Polydeoxyribonucleotide (PDRN), an adenosine A2A agonist, has positive effects on male reproductive system. We investigated the effects of PDRN on the morphological and functional changes induced by Cd in mice testes. Adult Swiss mice were divided into four groups: controls administered with 0.9% NaCl (1 ml/kg, i.p., daily) or with PDRN (8 mg/kg, i.p. daily), animals challenged with Cd chloride (CdCl2; 2 mg/kg, i.p, daily) and animals challenged with CdCl2 (2 mg/kg, i.p., daily) and treated with PDRN (8 mg/kg, i.p., daily). Experiments lasted 14 days. Testes were processed for biochemical, structural, and ultrastructural evaluation and hormones were assayed in serum. CdCl2 increased pERK 1/2 expression and Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels; it decreased testosterone (TE) and inhibin-B levels and induced structural damages in extratubular compartment and in seminiferous epithelium, with ultrastructural features of BTB disruption. Many TUNEL-positive germ cells were present. CdCl2 increased tubular TGF-β3 immunoreactivity and reduced claudin-11, occludin, and N-cadherin immunoreactivity. PDRN administration reduced pERK 1/2 expression, FSH, and LH levels; it increased TE and inhibin-B levels, ameliorated germinal epithelium changes and protected BTB ultrastructure. Few TUNEL-positive germ cells were present and the extratubular compartment was preserved. Furthermore, PDRN decreased TGF-β3 immunoreactivity and enhanced claudin-11, occludin, and N-cadherin immunoreactivity. We demonstrate a protective effect of PDRN on Cd-induced damages of BTB and suggest that PDRN may play an important role against Cd, particularly against its harmful effects on gametogenesis. PMID:28119612

  1. Phospholipase A2 inhibits cisplatin-induced acute kidney injury by modulating regulatory T cells by the CD206 mannose receptor.

    PubMed

    Kim, Hyunseong; Lee, Hyojung; Lee, Gihyun; Jang, Hyunil; Kim, Sung-Su; Yoon, Heera; Kang, Geun-Hyung; Hwang, Deok-Sang; Kim, Sun Kwang; Chung, Hwan-Suck; Bae, Hyunsu

    2015-09-01

    Previously, we found that Foxp3-expressing CD4(+) regulatory T (Treg) cells attenuate cisplatin-induced acute kidney injury in mice and that bee venom and its constituent phospholipase A2 (PLA2) are capable of modulating Treg cells. Here we tested whether PLA2 could inhibit cisplatin-induced acute kidney injury. As a result of treatment with PLA2, the population of Treg cells was significantly increased, both in vivo and in vitro. PLA2-injected mice showed reduced levels of serum creatinine, blood urea nitrogen, renal tissue damage, and pro-inflammatory cytokine production upon cisplatin administration. These renoprotective effects were abolished by depletion of Treg cells. Furthermore, PLA2 bound to CD206 mannose receptors on dendritic cells, essential for the PLA2-mediated protective effects on renal dysfunction. Interestingly, PLA2 treatment increased the secretion of IL-10 in the kidney from normal mice. Foxp3(+)IL-10(+) cells and CD11c(+)IL-10(+) cells were increased by PLA2 treatment. The anticancer effects of repeated administrations of a low dose of cisplatin were not affected by PLA2 treatment in a tumor-bearing model. Thus, PLA2 may prevent inflammatory responses in cisplatin-induced acute kidney injury by modulating Treg cells and IL-10 through the CD206 mannose receptor.

  2. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits

    PubMed Central

    Hussain, Suleman; Davanger, Svend

    2015-01-01

    Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism. PMID:26488171

  3. Berberine inhibits cytosolic phospholipase A2 and protects against LPS-induced lung injury and lethality independent of the alpha2-adrenergic receptor in mice.

    PubMed

    Zhang, Hao-qing; Wang, Hua-dong; Lu, Da-xiang; Qi, Ren-bin; Wang, Yan-ping; Yan, Yu-xia; Fu, Yong-mei

    2008-05-01

    Acute lung injury is still a significant clinical problem having a high mortality rate despite significant advances in antimicrobial therapy and supportive care made in the past few years. Our previous study demonstrated that berberine (Ber) remarkably decreased mortality and attenuated the lung injury in mice challenged with LPS, but the mechanism behind this remains unclear. Here, we report that pretreatment with Ber significantly reduced pulmonary edema, neutrophil infiltration, and histopathological alterations; inhibited protein expression and phosphorylation of cytosolic phospholipase A2; and decreased thromboxane A2 release induced by LPS. Yohimbine, an alpha2-adrenergic receptor antagonist, did not antagonize these actions of Ber. Furthermore, pretreatment with Ber decreased TNF-alpha production and mortality in mice challenged with LPS, which were enhanced by yohimbine, and Ber combined with yohimbine also improved survival rate in mice subjected to cecal ligation and puncture. Taken together, these observations indicate that Ber attenuates LPS-induced lung injury by inhibiting TNF-alpha production and cytosolic phospholipase A2 expression and activation in an alpha2-adrenoceptor-independent manner. Berberine combined with yohimbine might provide an effective therapeutic approach to acute lung injury during sepsis.

  4. Downregulation of A(1) and A(2B) adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Mirandola, Prisco; Bonfatti, Alessandra; Fini, Sergio; Sensi, Alberto; Marci, Roberto; Varani, Katia; Borea, Pier Andrea; Vesce, Fortunato

    2012-11-01

    Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.

  5. Adenosine A2a receptor stimulation blocks development of nonalcoholic steatohepatitis in mice by multilevel inhibition of signals that cause immunolipotoxicity.

    PubMed

    Alchera, Elisa; Rolla, Simona; Imarisio, Chiara; Bardina, Valentina; Valente, Guido; Novelli, Francesco; Carini, Rita

    2016-12-06

    Lipotoxicity and immunoinflammation are associated with the evolution of steatosis toward nonalcoholic steatohepatitis (NASH). This study reports the ability of adenosine A2a receptor (A2aR) activation to inhibit NASH development by modulating the responses of CD4(+) T-helper (Th) cells to avoid an immuno-mediated potentiation of lipotoxicity. The effect of the A2aR agonist CGS21680 on immunoinflammatory signals, CD4(+)Th cell infiltration and immunolipotoxicity was analyzed in steatotic C57BL/6 mice fed with a methionine-choline-deficient (MCD) diet and in mouse hepatocytes exposed to palmitic acid (PA). CGS21680 inhibited NASH development in steatotic mice and decreased cytokines and chemokines involved in Th cell recruitment or polarization (namely CXCL10, CCL2, tumor necrosis factor alfa [TNFα], tumor growth factor [TGFβ], and IL-12). CGS21680 also reduced the expansion of Th17, Th22, and Th1 cells and increased the immunosuppressive activity of T regulatory cells. In PA-treated mice hepatocytes, CGS21680 inhibited the production of CXCL10, TNFα, TGFβ, IL-12, and CCL2; CGS21680 also prevented JNK-dependent lipotoxicity and its intensification by IL-17 or IL-17 plus IL-22 through Akt/PI3-kinase stimulation and inhibition of the negative regulator of PI3-kinase, (phosphatase and tensin homologue deleted from chromosome 10 (PTEN), which is upregulated by IL-17. In MCD livers, CGS21680 reduced JNK activation and PTEN expression and increased Akt phosphorylation. In conclusion, A2aR stimulation inhibited NASH development by reducing Th17 cell expansion and inhibiting the exacerbation of the IL-17-induced JNK-dependent lipotoxicity. These data promote the implementation of further studies to evaluate the potential clinical application of A2aR agonists that, by being able to function as both cytoprotective and immunomodulatory agents, could efficiently antagonize the multi-faced pathogenesis of NASH.

  6. Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2–DP1 receptor paracrine axis

    PubMed Central

    Taketomi, Yoshitaka; Ueno, Noriko; Kojima, Takumi; Sato, Hiroyasu; Murase, Remi; Yamamoto, Kei; Tanaka, Satoshi; Sakanaka, Mariko; Nakamura, Masanori; Nishito, Yasumasa; Kawana, Momoko; Kambe, Naotomo; Ikeda, Kazutaka; Taguchi, Ryo; Nakamizo, Satoshi; Kabashima, Kenji; Gelb, Michael H.; Arita, Makoto; Yokomizo, Takehiko; Nakamura, Motonao; Watanabe, Kikuko; Hirai, Hiroyuki; Nakamura, Masataka; Okayama, Yoshimichi; Ra, Chisei; Aritake, Kosuke; Urade, Yoshihiro; Morimoto, Kazushi; Sugimoto, Yukihiko; Shimizu, Takao; Narumiya, Shuh; Hara, Shuntaro; Murakami, Makoto

    2014-01-01

    Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell–deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS–ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3–L-PGDS–DP1 loop that drives mast cell maturation. PMID:23624557

  7. MicroRNA signatures predict dysregulated vitamin D receptor and calcium pathways status in limb girdle muscle dystrophies (LGMD) 2A/2B.

    PubMed

    Aguennouz, M; Lo Giudice, C; Licata, N; Rodolico, C; Musumeci, O; Fanin, M; Migliorato, A; Ragusa, M; Macaione, V; Di Giorgio, R M; Angelini, C; Toscano, A

    2016-08-01

    miRNA expression profile and predicted pathways involved in selected limb-girdle muscular dystrophy (LGMD)2A/2B patients were investigated. A total of 187 miRNAs were dysregulated in all patients, with six miRNAs showing opposite regulation in LGMD2A versus LGMD2B patients. Silico analysis evidence: (1) a cluster of the dysregulated miRNAs resulted primarily involved in inflammation and calcium metabolism, and (2) two genes predicted as controlled by calcium-assigned miRNAs (Vitamin D Receptor gene and Guanine Nucleotide Binding protein beta polypeptide 1gene) showed an evident upregulation in LGMD2B patients, in accordance with miRNA levels. Our data support alterations in calcium pathway status in LGMD 2A/B, suggesting myofibre calcium imbalance as a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd.

  8. Effect of adenosine A(2A) receptor antagonists and L-DOPA on hydroxyl radical, glutamate and dopamine in the striatum of 6-OHDA-treated rats.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna

    2012-02-01

    A(2A) adenosine receptor antagonists have been proposed as a new therapy of PD. Since oxidative stress plays an important role in the pathogenesis of PD, we studied the effect of the selective A(2A) adenosine receptor antagonists 8-(-3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on hydroxyl radical generation, and glutamate (GLU) and dopamine (DA) extracellular level using a microdialysis in the striatum of 6-OHDA-treated rats. CSC (1 mg/kg) and ZM 241385 (3 mg/kg) given repeatedly for 14 days decreased the production of hydroxyl radical and extracellular GLU level, both enhanced by prior 6-OHDA treatment in dialysates from the rat striatum. CSC and ZM 241385 did not affect DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) extracellular levels in the striatum of 6-OHDA-treated rats. L-DOPA (6 mg/kg) given twice daily for two weeks in the presence of benserazide (3 mg/kg) decreased striatal hydroxyl radical and glutamate extracellular level in 6-OHDA-treated rats. At the same time, L-DOPA slightly but significantly increased the extracellular levels of DOPAC and HVA. A combined repeated administration of L-DOPA and CSC or ZM 241385 did not change the effect of L-DOPA on hydroxyl radical production and glutamate extracellular level in spite of an enhancement of extracellular DA level by CSC and elevation of extracellular level of DOPAC and HVA by ZM 241385. The data suggest that the 6-OHDA-induced damage of nigrostriatal DA-terminals is related to oxidative stress and excessive release of glutamate. Administration of L-DOPA in combination with CSC or ZM 241385, by restoring striatal DA-glutamate balance, suppressed 6-OHDA-induced overproduction of hydroxyl radical.

  9. Inosine, an Endogenous Purine Nucleoside, Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor.

    PubMed

    Junqueira, Stella Célio; Dos Santos Coelho, Igor; Lieberknecht, Vicente; Cunha, Mauricio Peña; Calixto, João B; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares; Dutra, Rafael Cypriano

    2016-04-30

    Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes

  10. Extending David Horrobin's membrane phospholipid theory of schizophrenia: overactivity of cytosolic phospholipase A(2) in the brain is caused by overdrive of coupled serotonergic 5HT(2A/2C) receptors in response to stress.

    PubMed

    Eggers, Arnold E

    2012-12-01

    David Horrobin's membrane phospholipid theory of schizophrenia has held up well over time because his therapeutic prediction that dietary supplementation with eicosapentaenoic acid (EPA) would have a therapeutic effect has been partially verified and undergoes continued testing. In the final version of his theory, he hypothesized that there was hyperactivity of phosphoslipase A(2) (PLA(2)) or a related enzyme but did not explain how the hyperactivity came about. It is known that serotonergic 5HT(2A/2C) receptors are coupled to PLA(2), which hydrolyzes both arachidonic acid (AA) and EPA from diacylglycerides at the sn-2 position. In this paper, Horrobin's theory is combined with a previously published theory of chronic stress in which it was hypothesized that a disinhibited dorsal raphe nucleus, the principal nucleus of the serotonergic system, can organize the neuropathology of diseases such as migraine, hypertension, and the metabolic syndrome. The new or combined theory is that schizophrenia is a disease of chronic stress in which a disinhibited DRN causes widespread serotonergic overdrive in the cerebral cortex. This in turn causes overdrive of cPLA(2) and both central and peripheral depletion of AA and EPA. Because EPA is present in smaller amounts, it falls below threshold for maintaining an intracellular balance between AA-derived and EPA-derived second messenger cascades, which leads to abnormal patterns of neuronal firing. There are two causes of neuronal dysfunction: the disinhibited DRN and EPA depletion. Schizophrenia is statistically associated with metabolic syndrome, hypertension, and migraine because they form a cluster of diseases with similar pathophysiology. The theory provides an explanation for both the central and peripheral phospholipid abnormalities in schizophrenia. It also explains the role of stress in schizophrenia, elevated serum PLA(2) activity in schizophrenia, the relationship between untreated schizophrenia and metabolic syndrome

  11. N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors.

    PubMed

    Drabczyńska, Anna; Müller, Christa E; Karolak-Wojciechowska, Janina; Schumacher, Britta; Schiedel, Anke; Yuzlenko, Olga; Kieć-Kononowicz, Katarzyna

    2007-07-15

    Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.

  12. A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

    PubMed

    Fresco, Paula; Oliveira, Jorge M A; Kunc, Filip; Soares, Ana Sofia; Rocha-Pereira, Carolina; Gonçalves, Jorge; Diniz, Carmen

    2007-07-01

    This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G

  13. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  14. Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: a role for A2A receptors.

    PubMed

    Mecha, M; Feliú, A; Iñigo, P M; Mestre, L; Carrillo-Salinas, F J; Guaza, C

    2013-11-01

    Inflammation in the central nervous system (CNS) is a complex process that involves a multitude of molecules and effectors, and it requires the transmigration of blood leukocytes across the blood-brain barrier (BBB) and the activation of resident immune cells. Cannabidiol (CBD), a non-psychotropic cannabinoid constituent of Cannabis sativa, has potent anti-inflammatory and immunosuppressive properties. Yet, how this compound modifies the deleterious effects of inflammation in TMEV-induced demyelinating disease (TMEV-IDD) remains unknown. Using this viral model of multiple sclerosis (MS), we demonstrate that CBD decreases the transmigration of blood leukocytes by downregulating the expression of vascular cell adhesion molecule-1 (VCAM-1), chemokines (CCL2 and CCL5) and the proinflammatory cytokine IL-1β, as well as by attenuating the activation of microglia. Moreover, CBD administration at the time of viral infection exerts long-lasting effects, ameliorating motor deficits in the chronic phase of the disease in conjunction with reduced microglial activation and pro-inflammatory cytokine production. Adenosine A2A receptors participate in some of the anti-inflammatory effects of CBD, as the A2A antagonist ZM241385 partially blocks the protective effects of CBD in the initial stages of inflammation. Together, our findings highlight the anti-inflammatory effects of CBD in this viral model of MS and demonstrate the significant therapeutic potential of this compound for the treatment of pathologies with an inflammatory component.

  15. Cytosolic phospholipase A2 is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: characterization of the transducing mechanism.

    PubMed Central

    Bayon, Y; Hernandez, M; Alonso, A; Nuñez, L; Garcia-Sancho, J; Leslie, C; Sanchez Crespo, M; Nieto, M L

    1997-01-01

    The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization. PMID:9173894

  16. Induction of progesterone receptor A form attenuates the induction of cytosolic phospholipase A2alpha expression by cortisol in human amnion fibroblasts.

    PubMed

    Guo, Chunming; Ni, Xiaotian; Zhu, Ping; Li, Wenjiao; Zhu, Xiaoou; Sun, Kang

    2010-05-01

    Cytosolic phospholipase A2alpha (cPLA(2alpha), now known as PLA2G4A) is the enzyme catalyzing the formation of the rate-limiting substrate, arachidonic acid, for prostaglandin (PG) synthesis. The increasing expression of PLA2G4A toward term gestation in human amnion fibroblasts is believed to be the crucial event in parturition. Human amnion fibroblasts produce cortisol, progesterone and express glucocorticoid receptor (GR), progesterone receptor A (PGRA) form at term. The roles of progesterone and PGRA in the induction of PLA2G4A by cortisol via GR in the amnion fibroblasts remain largely unknown. Using cultured human term amnion fibroblasts, we found that cortisol induced the expression of PGRA, which was attenuated by inhibiting PG synthesis with indomethacin. Knockdown of PGRA expression or inhibition of endogenous progesterone production with trilostane significantly enhanced the induction of PLA2G4A by cortisol, whereas overexpression of PGRA attenuated the induction of PLA2G4A by cortisol. Although exogenous progesterone did not alter PLA2G4A expression under basal conditions, it attenuated cortisol-induced PLA2G4A expression at concentrations about tenfold higher, which might be achieved by competition with cortisol for GR. In conclusion, PGRA in the presence of endogenous progesterone is a transdominant repressor of the induction of PLA2G4A by cortisol. High level of progesterone may compete with cortisol for GR, thus further inhibiting the induction of PLA2G4A by cortisol. Moreover, increased PG synthesis by cortisol may feed back on the expression of PGRA leading to attenuation of cortisol-induced PLA2G4A expression. The above findings may be pertinent to the inconsistent effects of glucocorticoids on parturition in humans.

  17. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel.

    PubMed

    Dawood, Ban B; Lowe, Gillian C; Lordkipanidzé, Marie; Bem, Danai; Daly, Martina E; Makris, Mike; Mumford, Andrew; Wilde, Jonathan T; Watson, Steve P

    2012-12-13

    Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

  18. Combining selectivity and affinity predictions using an integrated Support Vector Machine (SVM) approach: An alternative tool to discriminate between the human adenosine A(2A) and A(3) receptor pyrazolo-triazolo-pyrimidine antagonists binding sites.

    PubMed

    Michielan, Lisa; Bolcato, Chiara; Federico, Stephanie; Cacciari, Barbara; Bacilieri, Magdalena; Klotz, Karl-Norbert; Kachler, Sonja; Pastorin, Giorgia; Cardin, Riccardo; Sperduti, Alessandro; Spalluto, Giampiero; Moro, Stefano

    2009-07-15

    G Protein-coupled receptors (GPCRs) selectivity is an important aspect of drug discovery process, and distinguishing between related receptor subtypes is often the key to therapeutic success. Nowadays, very few valuable computational tools are available for the prediction of receptor subtypes selectivity. In the present study, we present an alternative application of the Support Vector Machine (SVM) and Support Vector Regression (SVR) methodologies to simultaneously describe both A(2A)R versus A(3)R subtypes selectivity profile and the corresponding receptor binding affinities. We have implemented an integrated application of SVM-SVR approach, based on the use of our recently reported autocorrelated molecular descriptors encoding for the Molecular Electrostatic Potential (autoMEP), to simultaneously discriminate A(2A)R versus A(3)R antagonists and to predict their binding affinity to the corresponding receptor subtype of a large dataset of known pyrazolo-triazolo-pyrimidine analogs. To validate our approach, we have synthetized 51 new pyrazolo-triazolo-pyrimidine derivatives anticipating both A(2A)R/A(3)R subtypes selectivity and receptor binding affinity profiles.

  19. Down-regulation of adenosine A1 and A2A receptors in peripheral cells from idiopathic normal-pressure hydrocephalus patients.

    PubMed

    Casati, Martina; Arosio, Beatrice; Gussago, Cristina; Ferri, Evelyn; Magni, Lorenzo; Assolari, Lara; Scortichini, Valeria; Nani, Carolina; Rossi, Paolo Dionigi; Mari, Daniela

    2016-02-15

    Idiopathic normal-pressure hydrocephalus (iNPH) is a neurological disease that usually develops in the elderly. Natural history of iNPH is still unknown. It has been hypothesized that cerebrovascular diseases could have a role in etiology of chronic hydrocephalus and studies show an increased prevalence of cardiovascular diseases in iNPH patients. Moreover, evidences show a possible alteration of immune system in iNPH patients. Adenosine (Ado) is a metabolite produced in response to metabolic stress and injury. Adenosine and its receptors play an important role in vascular protection and in the modulation of inflammatory reactions and neuroinflammation. Our aim is to evaluate gene and protein expression of A1R and A2AR in the peripheral blood mononuclear cells (PBMCs) from iNPH patients compared to control subjects. We investigate if Ado system, that plays an important role in central nervous system, in vascular system, and also in inflammation, is involved in pathophysiology of iNPH disease. Our analysis showed that A1R mRNA levels and A1R density in PBMCs from iNPH patients were significantly lower than CT subjects (0.84 ± 0.12 and 2.42 ± 0.42, p<0.001 and 0.31 ± 0.02 and 0.42 ± 0.04, p=0.043; respectively). About A2AR, the gene expression in PBMCs was significantly lower in iNPH than CT (0.65 ± 0.09 and 1.5 ± 0.14, p<0.001) as well as there was a trend in protein expression: iNPH and CT (0.51 ± 0.05 and 0.62 ± 0.03; p=0.172). This preliminary study underlines the involvement of Ado system in iNPH disease whose pathophysiology is still unclear.

  20. Activation of the thromboxane A2 receptor by 8-isoprostane inhibits the pro-angiogenic effect of vascular endothelial growth factor in scleroderma

    PubMed Central

    Tsou, Pei-Suen; Amin, M. Asif; Campbell, Phil; Zakhem, George; Balogh, Beatrix; Edhayan, Gautam; Ohara, Ray A.; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.; Fox, David A.

    2015-01-01

    The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and ROCK pathway. In this study we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs), and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc conditioned media inhibited EC tube formation, while addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 were elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs while VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in blockade of 8-isoprostane induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway plays a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress, but also plays a significant role in the impaired angiogenesis that characterizes SSc. PMID:26288351

  1. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression.

    PubMed

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei; Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2014-06-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A(2B) adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA(-/-) and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.

  2. Molecular docking studies of 1-(substituted phenyl)-3-(naphtha [1, 2-d] thiazol-2-yl) urea/thiourea derivatives with human adenosine A(2A) receptor.

    PubMed

    Azam, Faizul; Prasad, Medapati Vijaya Vara; Thangavel, Neelaveni; Ali, Hamed Ismail

    2011-01-01

    Computational assessment of the binding interactions of drugs is an important component of computer-aided drug design paradigms. In this perspective, a set of 30 1-(substituted phenyl)-3-(naphtha[1, 2-d] thiazol-2-yl) urea/thiourea derivatives showing antiparkinsonian activity were docked into inhibitor binding cavity of human adenosine A(2A) receptor (AA2AR) to understand their mode of binding interactions in silico. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.483) between docking score and antiparkinsonian activity (in terms of % reduction in catalepsy score). Potent antiparkinsonian agents carried methoxy group in the phenyl ring, exhibited both hydrophilic and lipophilic interactions with lower energy of binding at the AA(2A)R. These molecular docking analyses should, in our view, contribute for further development of selective AA(2A)R antagonists for the treatment of Parkinson's disease.

  3. Molecular Docking and Prediction of Pharmacokinetic Properties of Dual Mechanism Drugs that Block MAO-B and Adenosine A(2A) Receptors for the Treatment of Parkinson's Disease.

    PubMed

    Azam, Faizul; Madi, Arwa M; Ali, Hamed I

    2012-07-01

    Monoamine oxidase B (MAO-B) inhibitory potential of adenosine A(2A) receptor (AA(2A)R) antagonists has raised the possibility of designing dual-target-directed drugs that may provide enhanced symptomatic relief and that may also slow the progression of Parkinson's disease (PD) by protecting against further neurodegeneration. To explain the dual inhibition of MAO-B and AA(2A)R at the molecular level, molecular docking technique was employed. Lamarckian genetic algorithm methodology was used for flexible ligand docking studies. A good correlation (R(2)= 0.524 and 0.627 for MAO-B and AA(2A)R, respectively) was established between docking predicted and experimental K(i) values, which confirms that the molecular docking approach is reliable to study the mechanism of dual interaction of caffeinyl analogs with MAO-B and AA(2A)R. Parameters for Lipinski's "Rule-of-Five" were also calculated to estimate the pharmacokinetic properties of dual-target-directed drugs where both MAO-B inhibition and AA(2A)R antagonism exhibited a positive correlation with calculated LogP having a correlation coefficient R(2) of 0.535 and 0.607, respectively. These results provide some beneficial clues in structural modification for designing new inhibitors as dual-target-directed drugs with desired pharmacokinetic properties for the treatment of PD.

  4. Membranous nephropathy as a manifestation of graft-versus-host disease: association with HLA antigen typing, phospholipase A2 receptor, and C4d.

    PubMed

    Byrne-Dugan, Cathryn J; Collins, A Bernard; Lam, Albert Q; Batal, Ibrahim

    2014-12-01

    Glomerulopathy is an uncommon but increasingly recognized complication of hematopoietic cell transplantation. It typically manifests as membranous nephropathy, less commonly as minimal change disease, and rarely as proliferative glomerulonephritis. There is evidence to suggest that these glomerulopathies might represent manifestations of chronic graft-versus-host disease. In this report, we focus on membranous nephropathy as the most common form of glomerulopathy after hematopoietic cell transplantation. We present a case of membranous nephropathy that developed 483 days post-allogeneic hematopoietic stem cell transplantation in a patient with a history of acute graft-versus-host disease. We also share our experience with 4 other cases of membranous nephropathy occurring after allogeneic hematopoietic stem cell transplantation. Clinicopathologic correlates, including the association with graft-versus-host-disease, HLA antigen typing, glomerular deposition of immunoglobulin G (IgG) subclasses, subepithelial colocalization of IgG deposits with phospholipase A2 receptor staining, C4d deposition along the peritubular capillaries, and treatment, are discussed with references to the literature.

  5. Adenosine Type A2A Receptor in Peripheral Cell from Patients with Alzheimer's Disease, Vascular Dementia, and Idiopathic Normal Pressure Hydrocephalus: A New/Old Potential Target.

    PubMed

    Arosio, Beatrice; Casati, Martina; Gussago, Cristina; Ferri, Evelyn; Abbate, Carlo; Scortichini, Valeria; Colombo, Elena; Rossi, Paolo Dionigi; Mari, Daniela

    2016-09-06

    As the European population gets older, the incidence of neurological disorders increases with significant impact on social costs. Despite differences in disease etiology, several brain disorders in the elderly (e.g., Alzheimer's disease, vascular dementia, normal pressure hydrocephalus) share dementia as a common clinical feature. The current treatment for the majority of these diseases is merely symptomatic and does not modify the course of the illness. Symptoms of normal pressure hydrocephalus are the only ones that can be modified if they are recognized in time and treated appropriately. Therefore, an important clinical strategy may be disclosed by pathogenic pathways that can be modified and to find drugs that can slow down or even arrest disease progression. Possibly a way to answer this question could be by re-examining all the molecules which have so far succeeded in improving many aspects of cognitive deterioration in some neurodegenerative conditions, that were not considered because of controversial opinions. The main purpose of this summary is to further substantiate the hypothesis that the pathway of adenosine type A2A receptor could be used as a potential target to develop new/old therapeutic strategies.

  6. Mechanism of inhibition of GluA2 AMPA receptor channel opening by 2,3-benzodiazepine derivatives: functional consequences of replacing a 7,8-methylenedioxy with a 7,8-ethylenedioxy moiety.

    PubMed

    Qneibi, Mohammad S; Micale, Nicola; Grasso, Silvana; Niu, Li

    2012-02-28

    2,3-Benzodiazepine (2,3-BDZ) compounds are a group of AMPA receptor inhibitors and are drug candidates for treating neurological diseases involving excessive AMPA receptor activity. We investigated the mechanism by which GluA2Q(flip) receptor channel opening is inhibited by two 2,3-BDZ derivatives, i.e., 1-(4-aminophenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (2,3-BDZ-11-2) and its 1-(4-amino-3-chlorophenyl) analogue (2,3-BDZ-11-4). Both compounds have a 7,8-ethylenedioxy moiety instead of the 7,8-methylenedioxy feature present in the structure of GYKI 52466, the prototypic 2,3-BDZ compound. Using a laser-pulse photolysis approach with a time resolution of ~60 μs and a rapid solution flow technique, we characterized the effect of the two compounds on the channel opening process of the homomeric GluA2Q(flip) receptor. We found that both 2,3-BDZ-11-2 and 2,3-BDZ-11-4 are noncompetitive inhibitors with specificity for the closed-channel conformation of the GluA2Q(flip) receptor. However, 2,3-BDZ-11-4 is ~10-fold stronger, defined by its inhibition constant for the closed-channel conformation (i.e., K(I) = 2 μM), than 2,3-BDZ-11-2. From double-inhibitor experiments, we determined that both compounds bind to the same site, but this site is different from two other known, noncompetitive binding sites on the GluA2Q(flip) receptor previously reported. Our results provide both mechanistic clues to improve our understanding of AMPA receptor regulation and a structure-activity relationship for designing more potent 2,3-BDZ compounds with predictable properties for this new noncompetitive site.

  7. Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

    USGS Publications Warehouse

    Strobl-Mazzulla, P. H.; Lethimonier, C.; Gueguen, M.M.; Karube, M.; Fernandino, J.I.; Yoshizaki, G.; Patino, R.; Strussmann, C.A.; Kah, O.; Somoza, G.M.

    2008-01-01

    Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, ?? and ??, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor ??, but not ??, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate. ?? 2008 Elsevier Inc.

  8. Leishmania donovani-Induced Prostaglandin E2 Generation Is Critically Dependent on Host Toll-Like Receptor 2–Cytosolic Phospholipase A2 Signaling

    PubMed Central

    Bhattacharjee, Amrita; Majumder, Saikat; Das, Shibali; Ghosh, Sweta; Biswas, Satabdi

    2016-01-01

    Visceral leishmaniasis (VL) is the second-largest parasitic killer disease after malaria. During VL, the protozoan Leishmania donovani induces prostaglandin E2 (PGE2) generation within host macrophages to aid parasite survival. PGE2 significantly influences leishmanial pathogenesis, as L. donovani proliferation is known to be attenuated in PGE2-inhibited macrophages. Here, we report for the first time that signaling via macrophage Toll-like receptor 2 (TLR2) plays an instrumental role in inducing PGE2 release from L. donovani-infected macrophages. This signaling cascade, mediated via the TLR2–phosphatidylinositol 3-kinase (PI3K)–phospholipase C (PLC) signaling pathway, was found to be indispensable for activation of two major enzymes required for PGE2 generation: cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (Cox2). Inhibition of cPLA2, but not secreted phospholipase A2 (sPLA2) or calcium-independent phospholipase A2 (iPLA2), arrested L. donovani infection. During infection, cPLA2 activity increased >7-fold in a calcium-dependent and extracellular signal-regulated kinase (ERK)-dependent manner, indicating that elevation of intracellular calcium and ERK-mediated phosphorylation was necessary for L. donovani-induced cPLA2 activation. For transcriptional upregulation of cyclooxygenase 2, activation of the calcium-calcineurin-nuclear factor of activated T cells (NFAT) signaling was required in addition to the TLR2-PI3K-PLC pathway. Detailed studies by site-directed mutagenesis of potential NFAT binding sites and chromatin immunoprecipitation (ChIP) analysis revealed that the binding of macrophage NFATc2, at the −73/−77 site on the cox2 promoter, induced L. donovani-driven cox2 transcriptional activation. Collectively, these findings highlight the contribution of TLR2 downstream signaling toward activation of cPLA2 and Cox2 and illustrate how the TLR2-PI3K-PLC pathway acts in a concerted manner with calcium-calcineurin-NFATc2 signaling to modulate PGE2

  9. Nicotine and ethanol activate protein kinase A synergistically via G(i) betagamma subunits in nucleus accumbens/ventral tegmental cocultures: the role of dopamine D(1)/D(2) and adenosine A(2A) receptors.

    PubMed

    Inoue, Yuichiro; Yao, Lina; Hopf, F Woodward; Fan, Peidong; Jiang, Zhan; Bonci, Antonello; Diamond, Ivan

    2007-07-01

    Tobacco and alcohol are the most commonly used drugs of abuse and show the most serious comorbidity. The mesolimbic dopamine system contributes significantly to nicotine and ethanol reinforcement, but the underlying cellular signaling mechanisms are poorly understood. Nicotinic acetylcholine (nACh) receptors are highly expressed on ventral tegmental area (VTA) dopamine neurons, with relatively low expression in nucleus accumbens (NAcb) neurons. Because dopamine receptors D(1) and D(2) are highly expressed on NAcb neurons, nicotine could influence NAcb neurons indirectly by activating VTA neurons to release dopamine in the NAcb. To investigate this possibility in vitro, we established primary cultures containing neurons from VTA or NAcb separately or in cocultures. Nicotine increased cAMP response element-mediated gene expression only in cocultures; this increase was blocked by nACh or dopamine D(1) or D(2) receptor antagonists. Furthermore, subthreshold concentrations of nicotine with ethanol increased gene expression in cocultures, and this increase was blocked by nACh, D(2) or adenosine A(2A) receptor antagonists, Gbetagamma or protein kinase A (PKA) inhibitors, and adenosine deaminase. These results suggest that nicotine activated VTA neurons, causing the release of dopamine, which in turn stimulated both D(1) and D(2) receptors on NAcb neurons. In addition, subthreshold concentrations of nicotine and ethanol in combination also activated NAcb neurons through synergy between D(2) and A(2A) receptors. These data provide a novel cellular mechanism, involving Gbetagamma subunits, A(2A) receptors, and PKA, whereby combined use of tobacco and alcohol could enhance the reinforcing effect in humans as well as facilitate long-term neuroadaptations, increasing the risk for developing coaddiction.

  10. Optimization of 6-Heterocyclic-2-(1H-pyrazol-1-yl)-N-(pyridin-2-yl)pyrimidin-4-amine as Potent Adenosine A2A Receptor Antagonists for the Treatment of Parkinson’s Disease

    PubMed Central

    2014-01-01

    Parkinson’s disease is a neurodegenerative disease characterized by the motor symptoms of bradykinesia, tremor, and rigidity. Current therapies are based mainly on dopaminergic replacement strategies by administration of either dopamine agonists or dopamine precursor levodopa (L-Dopa). These treatments provide symptomatic relief without slowing or stopping the disease progression, and long-term usage of these drugs is associated with diminished efficacy, motor fluctuation, and dyskinisia. Unfortunately, there had been few novel treatments developed in the past decades. Among nondopaminergic strategies for the treatment of Parkinson’s disease, antagonism of the adenosine A2A receptor has emerged to show great potential. Here we report the optimization of a new chemical scaffold, which achieved exceptional receptor binding affinity and ligand efficiency against adenosine A2A receptor. The leading compounds demonstrated excellent efficacy in the haloperidol induced catalepsy model for Parkinson’s disease. PMID:24922583

  11. Synthesis and in vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

    PubMed

    Vala, Christine; Morley, Thomas J; Zhang, Xuechun; Papin, Caroline; Tavares, Adriana Alexandre S; Lee, H Sharon; Constantinescu, Cristian; Barret, Olivier; Carroll, Vincent M; Baldwin, Ronald M; Tamagnan, Gilles D; Alagille, David

    2016-09-06

    Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain.

  12. Anti-Phospholipase A2 Receptor (PLA2R) Antibody and Glomerular PLA2R Expression in Japanese Patients with Membranous Nephropathy

    PubMed Central

    Tachibana, Shohei; Iseri, Ken; Saito, Tomohiro; Yamamoto, Yasutaka; Suzuki, Taihei; Wada, Yukihiro; Matsumoto, Kei; Shibata, Takanori

    2016-01-01

    The phospholipase A2 receptor (PLA2R) is the major target antigen (Ag) in idiopathic membranous nephropathy (IMN). Recently, several types of immunoassay systems for anti-PLA2R antibody (Ab) have been developed. However, the correlation of serum anti-PLA2R Abs and glomerular expression of PLA2R Ag, and their association with clinicopathological characteristics have yet to be proven in Japanese patients. We examined serum anti-PLA2R Abs by both ELISA and cell-based indirect immunofluorescence assay (CIIFA), and glomerular PLA2R expression by immunofluorescence (IF) in 59 biopsy-proven MN patients including IMN (n = 38) and secondary MN (SMN) (n = 21). In this study, anti-PLA2R Abs were present in 50% of IMN patients, but was absent in SMN patients. The concordance rate between ELISA and CIIFA was 100%. Serum IgG levels were significantly lower in anti-PLA2R Ab-positive patients. Serum albumin levels correlated inversely with serum anti-PLA2R Ab titers. The prevalence and intensity of glomerular staining for IgG4 by IF were significantly higher in anti-PLA2R Ab-positive patients than in -negative patients. Glomerular PLA2 Ag expression evaluated by IF was positive in 52.6% of IMN patients, but was absent in SMN patients. The concordance rate between the prevalence of glomerular PLA2R Ag expression and anti-PLA2R Ab was 84.2%. The prevalence of anti-PLA2R Abs measured by ELISA/CIIFA was equivalent to previous Japanese studies evaluated using Western blotting. These analyses showed an excellent specificity for the diagnosis of IMN, and anti-PLA2R positivity was associated with some clinicopathological features, especially glomerular IgG4-dominant deposition. PMID:27355365

  13. Pharmacokinetics and metabolism of [(14)C]-tozadenant (SYN-115), a novel A2a receptor antagonist ligand, in healthy volunteers.

    PubMed

    Mancel, Valérie; Mathy, François-Xavier; Boulanger, Pierre; English, Stephen; Croft, Marie; Kenney, Christopher; Knott, Tarra; Stockis, Armel; Bani, Massimo

    2016-09-02

    1. This phase-I study (NCT02240290) was designed to investigate the human absorption, disposition and mass balance of (14)C-tozadenant, a novel A2a receptor antagonist in clinical development for Parkinson s disease. 2. Six healthy male subjects received a single oral dose of tozadenant (240 mg containing 81.47 KBq of [(14)C]-tozadenant). Blood, urine and feces were collected over 14 days. Radioactivity was determined by liquid scintillation counting or accelerator mass spectrometry (AMS). Tozadenant and metabolites were characterized using HPLC-MS/MS and HPLC-AMS with fraction collection. 3. At 4 h, the Cmax of tozadenant was 1.74 μg/mL and AUC(0-t) 35.0 h μg/mL, t1/2 15 h, Vz/F 1.82 L/kg and CL/F 1.40 mL/min/kg. For total [(14)C] radioactivity, the Cmax was 2.29 μg eq/mL at 5 h post-dose and AUC(0-t) 43.9 h μg eq/mL. Unchanged tozadenant amounted to 93% of the radiocarbon AUC(0-48h). At 312 h post-dose, cumulative urinary and fecal excretion of radiocarbon reached 30.5% and 55.1% of the dose, respectively. Unchanged tozadenant reached 11% in urine and 12% of the dose in feces. Tozadenant was excreted as metabolites, including di-and mono-hydroxylated metabolites, N/O dealkylated metabolites, hydrated metabolites. 4. The only identified species circulating in plasma was unchanged tozadenant. Tozadenant was primarily excreted in urine and feces in the form of metabolites.

  14. Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

    SciTech Connect

    Burroughs, S.F.; Johnson, G.J. )

    1990-04-01

    beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of (14C)-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with (3H)-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist (3H)-U46619 and antagonist (3H)-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ((Ca2+)i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in (Ca2+)i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.

  15. Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.

    PubMed Central

    Currie, S; Roberts, E F; Spaethe, S M; Roehm, N W; Kramer, R M

    1994-01-01

    In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the

  16. Increased striatal adenosine A2A receptor levels is an early event in Parkinson's disease-related pathology and it is potentially regulated by miR-34b.

    PubMed

    Villar-Menéndez, Izaskun; Porta, Sílvia; Buira, Sandra P; Pereira-Veiga, Thais; Díaz-Sánchez, Sara; Albasanz, José Luis; Ferrer, Isidre; Martín, Mairena; Barrachina, Marta

    2014-09-01

    Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.

  17. The Inhibitory Effect of α/β-Hydrolase Domain-Containing 6 (ABHD6) on the Surface Targeting of GluA2- and GluA3-Containing AMPA Receptors

    PubMed Central

    Wei, Mengping; Jia, Moye; Zhang, Jian; Yu, Lulu; Zhao, Yunzhi; Chen, Yingqi; Ma, Yimeng; Zhang, Wei; Shi, Yun S.; Zhang, Chen

    2017-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) are major excitatory receptors that mediate fast neurotransmission in the mammalian brain. The surface expression of functional AMPARs is crucial for synaptic transmission and plasticity. AMPAR auxiliary subunits control the biosynthesis, membrane trafficking, and synaptic targeting of AMPARs. Our previous report showed that α/β-hydrolase domain-containing 6 (ABHD6), an auxiliary subunit for AMPARs, suppresses the membrane delivery and function of GluA1-containing receptors in both heterologous cells and neurons. However, it remained unclear whether ABHD6 affects the membrane trafficking of glutamate receptor subunits, GluA2 and GluA3. Here, we examine the effects of ABHD6 overexpression in HEK293T cells expressing GluA1, GluA2, GluA3, and stargazin, either alone or in combination. The results show that ABHD6 suppresses the glutamate-induced currents and the membrane expression of AMPARs when expressing GluA2 or GluA3 in the HEK293T cells. We generated a series of GluA2 and GluA3 C-terminal deletion constructs and confirm that the C-terminus of GluAs is required for ABHD6’s inhibitory effects on glutamate-induced currents and surface expression of GluAs. Meanwhile, our pull-down experiments reveal that ABHD6 binds to GluA1–3, and deletion of the C-terminal domain of GluAs abolishes this binding. These findings demonstrate that ABHD6 inhibits the AMPAR-mediated currents and its surface expression, independent of the type of AMPAR subunits, and this inhibitor’s effects are mediated through the binding with the GluAs C-terminal regions. PMID:28303090

  18. Identification of a 2-phenyl-substituted octahydrobenzo[f]quinoline as a dopamine D3 receptor-selective full agonist ligand

    PubMed Central

    Clark, Alia H.; McCorvy, John D.; Conley, Jason M.; Williams, Whitney K.; Bekkam, Markondaiah; Watts, Val J.

    2012-01-01

    This work describes the identification of a novel class of octahydrobenzo[f]quinolines as dopamine D3-selective full agonists. We developed a facile method that utilizes Suzuki coupling for easy incorporations of various substituted pendant rings into the scaffold. A small focused library of octahydrobenzo[f]quinolines 5 was synthesized, and these compounds demonstrated at least 14-fold D2-like selectivity over D1 in native porcine striatal tissue. Furthermore, n-propyl analog 5f was found to be a high affinity (ki = 1.1 nM) D3 dopamine full agonist with 145-fold selectivity over the D2 receptor and about 840-fold selectivity over the D1 receptor. 1. PMID:23018094

  19. Brain penetrant liver X receptor (LXR) modulators based on a 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole core.

    PubMed

    Tice, Colin M; Noto, Paul B; Fan, Kristi Yi; Zhao, Wei; Lotesta, Stephen D; Dong, Chengguo; Marcus, Andrew P; Zheng, Ya-Jun; Chen, Guozhou; Wu, Zhongren; Van Orden, Rebecca; Zhou, Jing; Bukhtiyarov, Yuri; Zhao, Yi; Lipinski, Kerri; Howard, Lamont; Guo, Joan; Kandpal, Geeta; Meng, Shi; Hardy, Andrew; Krosky, Paula; Gregg, Richard E; Leftheris, Katerina; McKeever, Brian M; Singh, Suresh B; Lala, Deepak; McGeehan, Gerard M; Zhuang, Linghang; Claremon, David A

    2016-10-15

    Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected.

  20. Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane (TX) A{sub 2} receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F{sub 2{alpha}} and 8-iso-PGE{sub 2} regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy PGF{sub 2{alpha}} (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1{alpha},2{beta}(Z),3{alpha},5{alpha}

  1. Intracoronary administration of streptokinase in a canine model. Disturbance in thromboxane A2-prostacyclin balance.

    PubMed

    Becker, G J; Kreipke, D L; Holden, R W; Dreesen, R G; Ryder, K; Petersen, B; Evan, A P; Bendick, P J; Franklin, T D; Klatte, E C

    1986-03-01

    The potential impact of local intracoronary infusion of streptokinase (SK) on vascular prostaglandin synthetic pathways was studied in a canine model. Control animals (n = 10) underwent left coronary artery (LCA) infusion of 50,000 units SK for 90 minutes; experimental animals (n = 10) underwent LCA infusion of normal saline. Plasma samples for radioimmunoassay (RIA) of prostacyclin (PGI-2) and thromboxane (TXA-2) were obtained from the coronary sinus (CS) as follows: one sample preinfusion, six samples during infusion, and three samples postinfusion in each animal. Comparisons between control and experimental plasma levels of PGI-2 and TXA-2 were made for each sampling time. The PGI-2 levels remained at or below the lower limits of detectability by RIA (the most sensitive assay available) in both control and experimental animals. TXA-2 levels were higher in experimental than in control animals at all sampling times, with the most significant differences occurring in samples 3 (after 30 minutes of infusion, .001 less than P less than .01), 4 (after 45 minutes of infusion, .05 less than P less than .10), and 5 (after 60 minutes of infusion, .02 less than P less than .05). We suggest (1) it is unlikely that any of the beneficial effects of coronary streptokinase infusions are PGI-2-mediated, (2) that the TXA-2 increases in our model may represent a pathophysiologic-biochemical correlate of previously identified morphologic evidence of endothelial damage in animals infused with fibrinolytic agents, and (3) that our findings may indicate that fibrinolytic infusions produce competing effects: lysis of thrombi and endothelial injury with TXA-2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Restricted access to standard or high fat chow alters sensitivity of rats to the 5-HT2A/2C receptor agonist 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM)

    PubMed Central

    Serafine, Katherine M.; France, Charles P.

    2017-01-01

    Feeding conditions can impact sensitivity to drugs acting on dopamine receptors; less is known about the impact of feeding conditions on the effects of drugs acting on serotonin (5-HT) receptors. This study examined the effects of feeding condition on sensitivity to the direct-acting 5-HT2A/2C receptor agonist 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM; 0.1–3.2 mg/kg) and the direct-acting dopamineD3/D2 receptor agonist quinpirole (0.0032–0.32 mg/kg). Male Sprague-Dawley rats had free access (11 weeks) followed by restricted access (6 weeks) to high (34.3%, n = 8) fat or standard (5.7% fat; n = 7) chow. Rats eating high fat chow became insulin resistant and gained more weight than rats eating standard chow. Free access to high fat chow did not alter sensitivity to DOM-induced head twitch but increased sensitivity to quinpirole-induced yawning. Restricting access to high fat or standard chow shifted the DOM-induced head twitch dose-response curve to the right and shifted the quinpirole-induced yawning dose-response curve downward in both groups of rats. Some drugs of abuse and many therapeutic drugs act on 5-HT and dopamine systems; these results demonstrate that feeding condition impacts sensitivity to drugs acting on these systems, thereby possibly impacting vulnerability to abuse as well as therapeutic effectiveness of drugs. PMID:24346289

  3. Concurrent agonism of adenosine A2B and glucocorticoid receptors in human airway epithelial cells cooperatively induces genes with anti-inflammatory potential: a novel approach to treat chronic obstructive pulmonary disease.

    PubMed

    Greer, Stephanie; Page, Cara W; Joshi, Taruna; Yan, Dong; Newton, Robert; Giembycz, Mark A

    2013-09-01

    Chronic obstructive pulmonary disease (COPD) is a neutrophilic inflammatory disorder that is weakly responsive to glucocorticoids. Identification of ways to enhance the anti-inflammatory activity of glucocorticoids is, therefore, a major research objective. Adenosine receptor agonists that target the A2B-receptor subtype are efficacious in several cell-based assays and preclinical models of inflammation. Accordingly, the present study was designed to determine if a selective A2B-receptor agonist, 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulphanyl]acetamide (Bay 60-6583), and a glucocorticoid, dexamethasone, in combination display putative anti-inflammatory activity that is superior to either drug alone. In BEAS-2B human airway epithelial cells stably transfected with cAMP-response element (CRE) and glucocorticoid response element (GRE) reporter constructs, Bay 60-6583 promoted CRE-dependent transcription and enhanced GRE-dependent transcription by an adenosine A2B-receptor-mediated mechanism that was associated with cAMP formation and abolished by an inhibitor of cAMP-dependent protein kinase. Analysis of the concentration-response relationship that described the enhancement of GRE-dependent transcription showed that Bay 60-6583 increased the magnitude of response without affecting the potency of dexamethasone. Bay 60-6583 and dexamethasone also induced a panel of genes that, collectively, could have benefit in COPD. These were categorized into genes that were induced in a positive cooperative manner (RGS2, p57(kip2)), an additive manner (TTP, BRL-1), or by Bay 60-6583 (CD200, CRISPLD2, SOCS3) or dexamethasone (GILZ) only. Thus, the gene induction "fingerprints" produced by Bay 60-6583 and dexamethasone, alone and in combination, were distinct. Collectively, through their actions on gene expression, an adenosine A2B-receptor agonist and a glucocorticoid administered together may have utility in the treatment of inflammatory disorders that

  4. Blockade of striatal adenosine A2A receptor reduces, through a presynaptic mechanism, quinolinic acid-induced excitotoxicity: possible relevance to neuroprotective interventions in neurodegenerative diseases of the striatum.

    PubMed

    Popoli, Patrizia; Pintor, Annita; Domenici, Maria Rosaria; Frank, Claudio; Tebano, Maria Teresa; Pèzzola, Antonella; Scarchilli, Laura; Quarta, Davide; Reggio, Rosaria; Malchiodi-Albedi, Fiorella; Falchi, Mario; Massotti, Marino

    2002-03-01

    The aim of the present study was to evaluate whether, and by means of which mechanisms, the adenosine A2A receptor antagonist SCH 58261 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine] exerted neuroprotective effects in a rat model of Huntington's disease. In a first set of experiments, SCH 58261 (0.01 and 1 mg/kg) was administered intraperitoneally to Wistar rats 20 min before the bilateral striatal injection of quinolinic acid (QA) (300 nmol/1 microl). SCH 58261 (0.01 but not 1 mg/kg, i.p.) did reduce significantly the effects of QA on motor activity, electroencephalographic changes, and striatal gliosis. Because QA acts by both increasing glutamate outflow and directly stimulating NMDA receptors, a second set of experiments was performed to evaluate whether SCH 58261 acted by preventing the presynaptic and/or the postsynaptic effects of QA. In microdialysis experiments in naive rats, striatal perfusion with QA (5 mm) enhanced glutamate levels by approximately 500%. Such an effect of QA was completely antagonized by pretreatment with SCH 58261 (0.01 but not 1 mg/kg, i.p.). In primary striatal cultures, bath application of QA (900 microm) significantly increased intracellular calcium levels, an effect prevented by the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. In this model, bath application of SCH 58261 (15-200 nm) tended to potentiate QA-induced calcium increase. We conclude the following: (1) the adenosine A2A receptor antagonist SCH 58261 has neuroprotective effects, although only at low doses, in an excitotoxic rat model of HD, and (2) the inhibition of QA-evoked glutamate outflow seems to be the major mechanism underlying the neuroprotective effects of SCH 58261.

  5. Effects of the adenosine A2A receptor antagonist SCH 58621 on cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor availability in a rat model of striatal neurodegeneration.

    PubMed

    Minghetti, Luisa; Greco, Anita; Potenza, Rosa Luisa; Pezzola, Antonella; Blum, David; Bantubungi, Kadiombo; Popoli, Patrizia

    2007-05-01

    Inhibition of adenosine A2A receptors (A2ARs) is neuroprotective in several experimental models of striatal diseases. However, the mechanisms elicited by A2AR blockade are only partially known, and critical aspects about the potential beneficial effects of A2AR antagonism in models of neurodegeneration still await elucidation. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitotoxicity obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons and prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the 2 brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain-derived neurotrophic factor levels. Because COX-2 activity has been linked to excitotoxic processes and because brain-derived neurotrophic factor depletion has been observed in mouse models as well as in patients with Huntington disease, we suggest that the final outcome of A2AR blockade (namely neuroprotection vs neurodegeneration) is likely to depend on the balance among its various and region-specific effects.

  6. An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity

    PubMed Central

    Li, Xiaohang; Wang, Xin; Zhou, Yue; Yang, Wenjuan; Chen, Ming; Zhao, Jian; Pei, Gang

    2016-01-01

    γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aβ which is associated with Alzheimer’s disease (AD). Here we identified that an anti-Parkinson’s disease drug, Istradefylline, could enhance Aβ generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aβ42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aβ production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or β-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aβ generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aβ generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS). PMID:27835671

  7. Role of enhanced glomerular synthesis of thromboxane A2 in progressive kidney disease.

    PubMed

    Salvati, P; Ferti, C; Ferrario, R G; Lamberti, E; Duzzi, L; Bianchi, G; Remuzzi, G; Perico, N; Benigni, A; Braidotti, P

    1990-09-01

    Normotensive rats of the Milan strain (MNS) spontaneously develop focal glomerulosclerosis. In order to explore the contribution of glomerular thromboxane (TX) A2 synthesis to the development of the disease, we have characterized the time course of renal functional and biochemical changes, and their modification by long-term treatment with a TX-synthase inhibitor. Oral administration (150 mg.kg-1 from 1 to 14 months of age) of FCE 22178 suppressed enhanced glomerular TXB2 production at all experimental times (mean inhibition 80%) and proteinuria (varying between 27.1 and 73.0%) while preserving renal blood flow and glomerular filtration rate. These effects of TX-synthase inhibition were seen in the absence of any statistically significant changes in systemic blood pressure. Moreover, FCE 22178 had no antihypertensive effects in hypertensive rats of the Milan strain (MHS) nor in spontaneously hypertensive rats (SHR). Treatment also prevented the age-related hypoalbuminemia and hyperlipidemia observed in control MNS and significantly (P less than 0.01) reduced glomerular histologic damage, as demonstrated by light microscopy studies and measurement of sclerotic area. We conclude that: 1) MNS rats provide an animal model of long-lasting proteinuria characterized by an age-related increase in glomerular TXB2 production paralleled by progressive loss of renal structural integrity and function and by a secondary dyslipidemia; 2) pharmacological inhibition of glomerular TX-synthase attenuates the structural as well as the functional expression of kidney disease, without a primary effect on systemic blood pressure. These data are suggestive of an important modulating role of TXA2 in the progression of MNS renal disease.

  8. The aryl hydrocarbon receptor-interacting protein (AIP) is required for dioxin-induced hepatotoxicity but not for the induction of the Cyp1a1 and Cyp1a2 genes.

    PubMed

    Nukaya, Manabu; Lin, Bernice C; Glover, Edward; Moran, Susan M; Kennedy, Gregory D; Bradfield, Christopher A

    2010-11-12

    The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.

  9. Stimulation of 5-HT1A, 5-HT1B, 5-HT2A/2C, 5-HT3 and 5-HT4 receptors or 5-HT uptake inhibition: short- and long-term memory.

    PubMed

    Meneses, Alfredo

    2007-11-22

    In order to determine whether short- (STM) and long-term memory (LTM) function in serial or parallel manner, serotonin (5-hydroxtryptamine, 5-HT) receptor agonists were tested in autoshaping task. Results show that control-vehicle animals were modestly but significantly mastering the autoshaping task as illustrated by memory scores between STM and LTM. Thus, post-training administration of 8-OHDPAT (agonist for 5-HT(1A/7) receptors) only at 0.250 and 0.500 mg/kg impaired both STM and LTM. CGS12066 (agonist for 5-HT(1B)) produced biphasic affects, at 5.0 mg/kg impaired STM but at 1.0 and 10.0 mg/kg, respectively, improved or impaired LTM. DOI (agonist for 5-HT(2A/2C) receptors) dose-dependently impaired STM and, at 10.0 mg/kg only impaired LTM. Both, STM and LTM were impaired by either mCPP (mainly agonist for 5-HT(2C) receptors) or mesulergine (mainly antagonist for 5-HT(2C) receptors) lower dose. The 5-HT(3) agonist mCPBG at 1.0 impaired STM and its higher dose impaired both STM and LTM. RS67333 (partial agonist for 5-HT(4) receptors), at 5.0 and 10.0 mg/kg facilitated both STM and LTM. The higher dose of fluoxetine (a 5-HT uptake inhibitor) improved both STM and LTM. Using as head-pokes during CS as an indirect measure of food-intake showed that of 30 memory changes, 21 of these were unrelated to the former. While some STM or LTM impairments can be attributed to decrements in food-intake, but not memory changes (either increase or decreases) produced by 8-OHDPAT, CGS12066, RS67333 or fluoxetine. Except for animals treated with DOI, mCPBG or fluoxetine, other groups treated with 5-HT agonists 6 h following autoshaping training showed similar LTM and unmodified CS-head-pokes scores.

  10. The receptor EP3 to PGE2: A rational target to prevent atherothrombosis without inducing bleeding.

    PubMed

    Mawhin, Marie-Anne; Tilly, Peggy; Fabre, Jean-Etienne

    2015-09-01

    The prostanoid E2 (PGE2) is known to modulate the aggregative response of platelets to their conventional agonists such as ADP, TXA2, thrombin or collagen. Through the activation of its receptor EP3, PGE2 sensitizes platelets to their agonists but also inhibits them through its two other receptors, EP2 and EP4. In mice, the net result of these opposed actions is the EP3-mediated potentiation of platelet aggregation and the in vivo aggravation of murine atherothrombosis. Since the pathway PGE2/EP3 is not involved in murine hemostasis, we propose a "platelet EP3 paradigm" to describe this apparently paradoxical association between the facilitating impact on atherothrombosis and the unaltered hemostasis. Consistent with this paradigm, a drug blocking EP3 dramatically decreased atherothrombosis without inducing bleeding in mice. In humans, several studies did not agree on the effect of PGE2 on platelets. Reinterpreting these data with the notion of "potentiation window" and taking the platelet initial cAMP level into account reconciled these inconsistent results. Thereby, the in vitro potentiating effect of PGE2 on human platelets becomes clear. In addition, the EP3 blocking drug DG-041 abrogated the potentiating effect of PGE2 in whole human blood but did not prolong bleeding times in volunteers. Thus, the murine "platelet EP3 paradigm" would apply to humans if the aggravating role of PGE2 on atherothrombosis is shown in patients. Therefore, testing an EP3 blocker in a phase III trial would be of high interest to fulfill the unmet medical need which is to control atherothrombosis without impacting hemostasis and thus to improve the prevention of myocardial infarction.

  11. The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors.

    PubMed

    Tian, Yikui; Marshall, Melissa; French, Brent A; Linden, Joel; Yang, Zequan

    2015-03-01

    Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 μg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.

  12. Effects of central activation of serotonin 5-HT2A/2C or dopamine D2/3 receptors on the acute and repeated effects of clozapine in the conditioned avoidance response test

    PubMed Central

    Feng, Min; Gao, Jun; Sui, Nan; Li, Ming

    2014-01-01

    Rationale: Acute administration of clozapine (a gold standard of atypical antipsychotics) disrupts avoidance response in rodents, while repeated administration often causes a tolerance effect. Objective: The present study investigated the neuroanatomical basis and receptor mechanisms of acute and repeated effects of clozapine treatment in the conditioned avoidance response test in male Sprague-Dawley rats. Methods: DOI (2,5-dimethoxy-4-iodo-amphetamine, a preferential 5-HT2A/2C agonist) or quinpirole (a preferential dopamine D2/3 agonist) was microinjected into the medial prefrontal cortex (mPFC) or nucleus accumbens shell (NAs), and their effects on the acute and long-term avoidance-disruptive effect of clozapine were tested. Results: Intra-mPFC microinjection of quinpirole enhanced the acute avoidance disruptive effect of clozapine (10 mg/kg, sc), while DOI microinjections reduced it marginally. Repeated administration of clozapine (10 mg/kg, sc) daily for 5 days caused a progressive decrease in its inhibition of avoidance responding, indicating tolerance development. Intra-mPFC microinjection of DOI at 25.0 (but not 5.0) μg/side during this period completely abolished the expression of clozapine tolerance. This was indicated by the finding that clozapine-treated rats centrally infused with 25.0 μg/side DOI did not show higher levels of avoidance responses than the vehicle-treated rats in the clozapine challenge test. Microinjection of DOI into the mPFC immediately before the challenge test also decreased the expression of clozapine tolerance. Conclusions: Acute behavioral effect of clozapine can be enhanced by activation of the D2/3 receptors in the mPFC. Clozapine tolerance expression relies on the neuroplasticity initiated by its antagonist action against 5-HT2A/2C receptors in the mPFC. PMID:25288514

  13. Serum levels of osteoprotegerin and receptor activator of nuclear factor -κB ligand in children with early juvenile idiopathic arthritis: a 2-year prospective controlled study

    PubMed Central

    2010-01-01

    Background The clinical relevance of observations of serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor -κB ligand (RANKL) in juvenile idiopathic arthritis (JIA) is not clear. To elucidate the potential role of OPG and RANKL in JIA we determined serum levels of OPG and RANKL in patients with early JIA compared to healthy children, and prospectively explored changes in relation to radiographic score, bone and lean mass, severity of the disease, and treatment. Methods Ninety children with early oligoarticular or polyarticular JIA (ages 6-18 years; mean disease duration 19.4 months) and 90 healthy children individually matched for age, sex, race, and county of residence, were examined at baseline and 2-year follow-up. OPG and RANKL were quantified by enzyme-immunoassay. Data were analyzed with the use of t-tests, ANOVA, and multiple regression analyses. Results Serum OPG was significantly lower in patients than controls at baseline, and there was a trend towards higher RANKL and a lower OPG/RANKL ratio. Patients with polyarthritis had significantly higher increments in RANKL from baseline to follow-up, compared to patients with oligoarthritis. RANKL was a significant negative predictor for increments in total body lean mass. Patients who were receiving corticosteroids (CS) or disease-modifying antirheumatic drugs (DMARDs) at follow-up had higher OPG/RANKL ratio compared with patients who did not receive this medication. Conclusions The data supports that levels of OPG are lower in patients with JIA compared to healthy children, and higher levels of RANKL is associated with more serious disease. RANKL was a significant negative predictor of lean mass in patients with JIA. The OPG/RANKL ratio was higher in patients on DMARDs or CS treatment. PMID:21134287

  14. Adenosine A2A Receptor Gene Knockout Prevents l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia by Downregulation of Striatal GAD67 in 6-OHDA-Lesioned Parkinson’s Mice

    PubMed Central

    Yin, Su-bing; Zhang, Xiao-guang; Chen, Shuang; Yang, Wen-ting; Zheng, Xia-wei; Zheng, Guo-qing

    2017-01-01

    l-3,4-Dihydroxyphenylalanine (l-DOPA) remains the primary pharmacological agent for the symptomatic treatment of Parkinson’s disease (PD). However, the development of l-DOPA-induced dyskinesia (LID) limits the long-term use of l-DOPA for PD patients. Some data have reported that adenosine A2A receptor (A2AR) antagonists prevented LID in animal model of PD. However, the mechanism in which adenosine A2AR blockade alleviates the symptoms of LID has not been fully clarified. Here, we determined to knock out (KO) the gene of A2AR and explored the possible underlying mechanisms implicated in development of LID in a mouse model of PD. A2AR gene KO mice were unilaterally injected into the striatum with 6-hydroxydopamine (6-OHDA) in order to damage dopamine neurons on one side of the brain. 6-OHDA-lesioned mice were then injected once daily for 21 days with l-DOPA. Abnormal involuntary movements (AIMs) were evaluated on days 3, 8, 13, and 18 after l-DOPA administration, and real-time polymerase chain reaction and immunohistochemistry for glutamic acid decarboxylase (GAD) 65 and GAD67 were performed. We found that A2AR gene KO was effective in reducing AIM scores and accompanied with decrease of striatal GAD67, rather than GAD65. These results demonstrated that the possible mechanism involved in alleviation of AIM symptoms by A2AR gene KO might be through reducing the expression of striatal GAD67. PMID:28377741

  15. Pulsed electromagnetic fields increased the anti-inflammatory effect of A₂A and A₃ adenosine receptors in human T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts.

    PubMed

    Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Gessi, Stefania; Merighi, Stefania; Setti, Stefania; Cadossi, Ruggero; Goldring, Mary B; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders.

  16. 3D-QSAR methods on the basis of ligand-receptor complexes. Application of COMBINE and GRID/GOLPE methodologies to a series of CYP1A2 ligands.

    PubMed

    Lozano, J J; Pastor, M; Cruciani, G; Gaedt, K; Centeno, N B; Gago, F; Sanz, F

    2000-05-01

    Many heterocyclic amines (HCA) present in cooked food exert a genotoxic activity when they are metabolised (N-oxidated) by the human cytochrome P450 1A2 (CYP1A2h). In order to rationalize the observed differences in activity of this enzyme on a series of 12 HCA, 3D-QSAR methods were applied on the basis of models of HCA-CYP1A2h complexes. The CYP1A2h enzyme model has been previously reported and was built by homology modeling based on cytochrome P450 BM3. The complexes were automatically generated applying the AUTODOCK software and refined using AMBER. A COMBINE analysis on the complexes identified the most important enzyme-ligand interactions that account for the differences in activity within the series. A GRID/GOLPE analysis was then performed on just the ligands, in the conformations and orientations found in the modeled complexes. The results from both methods were concordant and confirmed the advantages of incorporating structural information from series of ligand-receptor complexes into 3D-QSAR methodologies.

  17. 3D-QSAR methods on the basis of ligand-receptor complexes. Application of COMBINE and GRID/GOLPE methodologies to a series of CYP1A2 ligands

    NASA Astrophysics Data System (ADS)

    Lozano, Juan José; Pastor, Manuel; Cruciani, Gabriele; Gaedt, Katrin; Centeno, Nuria B.; Gago, Federico; Sanz, Ferran

    2000-05-01

    Many heterocyclic amines (HCA) present in cooked food exert a genotoxic activity when they are metabolised (N-oxidated) by the human cytochrome P450 1A2 (CYP1A2h). In order to rationalize the observed differences in activity of this enzyme on a series of 12 HCA, 3D-QSAR methods were applied on the basis of models of HCA-CYP1A2h complexes. The CYP1A2h enzyme model has been previously reported and was built by homology modeling based on cytochrome P450 BM3. The complexes were automatically generated applying the AUTODOCK software and refined using AMBER. A COMBINE analysis on the complexes identified the most important enzyme-ligand interactions that account for the differences in activity within the series. A GRID/GOLPE analysis was then performed on just the ligands, in the conformations and orientations found in the modeled complexes. The results from both methods were concordant and confirmed the advantages of incorporating structural information from series of ligand-receptor complexes into 3D-QSAR methodologies.

  18. The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles.

    PubMed

    Squarcialupi, Lucia; Betti, Marco; Catarzi, Daniela; Varano, Flavia; Falsini, Matteo; Ravani, Annalisa; Pasquini, Silvia; Vincenzi, Fabrizio; Salmaso, Veronica; Sturlese, Mattia; Varani, Katia; Moro, Stefano; Colotta, Vittoria

    2017-12-01

    New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N(4)-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].

  19. TSU-16, (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a potent activator of aryl hydrocarbon receptor and increases CYP1A1 and CYP1A2 expression in human hepatocytes.

    PubMed

    Matsuoka-Kawano, Kazuaki; Yoshinari, Kouichi; Nagayama, Sekio; Yamazoe, Yasushi

    2010-04-15

    (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (TSU-16), is a potent anti-angiogenic agent that inhibits the tyrosine kinase of vascular endothelial growth factor receptor-2. In clinical trials with daily or twice weekly intravenous administration of TSU-16, its increased clearance was observed. To understand the mechanism underlying this observation, we have investigated the TSU-16-mediated regulation of cytochrome P450 expression. In human hepatocytes, TSU-16 increased mRNA levels of CYP1A1 and CYP1A2, but not CYP2B6 and CYP3A4. The extent of increase and profiles of the time-dependent changes in CYP1A1 and CYP1A2 mRNA levels after TSU-16 treatment were similar to those after treatment with 3-methylcholanthrene (3MC), a well-known activator of the aryl hydrocarbon receptor (AhR). In reporter assays using a plasmid construct that contained the human CYP1A1 5'-flanking region including the region crucial for the AhR-dependent transcription of both human CYP1A1 and CYP1A2, TSU-16 treatment increased reporter activities to an extent similar to that obtained with 3MC. Treatment of HepG2 cells and human hepatocytes with AhR-targeting siRNA suppressed the increase in both mRNA levels and CYP1A activities after treatment with TSU-16 as well as after that with omeprazole or 3MC. TSU-16 also time-dependently reduced cellular AhR protein levels in HepG2 cells to a similar extent with 3MC treatment. Furthermore, we demonstrated that unlabeled TSU-16 and 3MC but not omeprazole completely inhibited the specific binding of [(3)H]-3MC to mouse Hepa1c1c7 cytosol, suggesting TSU-16 as an AhR ligand. In conclusion, our present results suggest that TSU-16 binds to and activates AhR to enhance the expression of both human CYP1A1 and CYP1A2. Because TSU-16 is metabolized mainly by CYP1A2, its increased clearance after repeated dosing may be attributed to the enhanced expression of hepatic CYP1A2.

  20. The comparative effects of diethyldithiocarbamate-copper complex with established proteasome inhibitors on expression levels of CYP1A2/3A4 and their master regulators, aryl hydrocarbon and pregnane X receptor in primary cultures of human hepatocytes.

    PubMed

    Vrzal, Radim; Dvorak, Zdenek

    2016-12-01

    In the recent years, a therapeutic potential of disulfiram (Antabuse) complex with copper, as an anticancer drug, was recognized towards several cancer cell lines. The proteasome was suggested as one of the cellular targets for this compound. As the therapeutic use of diethyldithiocarbamate-copper complex (CuET) is expected to increase, it is of great interest to know whether this compound may be the source of drug-drug interactions via the induction of biotransformation enzymes, especially cytochromes P450 (CYPs). To this purpose, we examined the effect of CuET and compared it with typical inducers (rifampicin and dioxin) of CYPs and with well-established proteasome inhibitors (MG132 and bortezomib). Diethyldithiocarbamate-copper complex revealed inconsistent and rather modulatory effect on the expression of CYP1A2 and CYP3A4 in several cultures of human hepatocytes. Moreover, it was able to cause neither ubiquitin accumulation nor significant and dose-dependent inhibition of proteasome activity. It had no effect on essential transcription factors involved in regulation of selected CYPs, aryl hydrocarbon (AhR) nor pregnane X receptor (PXR). However, the AhR protein was increased in majority of examined hepatocyte cultures. The main finding of this study is that: (i) disulfiram-copper complex is not the cause of drug-drug interactions via CYP1A2/3A4 induction; (ii) proteasome inhibitors may have different impact on studied parameters in given in vitro system.

  1. Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes

    PubMed Central

    Ji, X.; Maxwell, J. T.; Mignery, G. A.; Samarel, A. M.; Lipsius, S. L.

    2016-01-01

    We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the

  2. Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn

    PubMed Central

    Majed, Batoule H.

    2012-01-01

    Prostacyclin (PGI2) is a member of the prostanoid group of eicosanoids that regulate homeostasis, hemostasis, smooth muscle function and inflammation. Prostanoids are derived from arachidonic acid by the sequential actions of phospholipase A2, cyclooxygenase (COX), and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX1 and COX2, that differ in structure, tissue distribution, subcellular localization, and function. COX1 is largely constitutively expressed, whereas COX2 is induced at sites of inflammation and vascular injury. PGI2 is produced by endothelial cells and influences many cardiovascular processes. PGI2 acts mainly on the prostacyclin (IP) receptor, but because of receptor homology, PGI2 analogs such as iloprost may act on other prostanoid receptors with variable affinities. PGI2/IP interaction stimulates G protein-coupled increase in cAMP and protein kinase A, resulting in decreased [Ca2+]i, and could also cause inhibition of Rho kinase, leading to vascular smooth muscle relaxation. In addition, PGI2 intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription. PGI2 counteracts the vasoconstrictor and platelet aggregation effects of thromboxane A2 (TXA2), and both prostanoids create an important balance in cardiovascular homeostasis. The PGI2/TXA2 balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. A decrease in PGI2/TXA2 ratio in the maternal, fetal, and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction, and persistent pulmonary hypertension of the newborn (PPHN), respectively. On the other hand, increased PGI2 activity may contribute to patent ductus arteriosus (PDA) and intraventricular hemorrhage in premature newborns. These observations have raised interest in the use of COX inhibitors and PGI2 analogs in the management of pregnancy-associated and neonatal

  3. Food extracts consumed in Mediterranean countries and East Asia reduce protein concentrations of androgen receptor, phospho-protein kinase B, and phospho-cytosolic phospholipase A(2)alpha in human prostate cancer cells.

    PubMed

    Singh, Jaskirat; Xie, Chanlu; Yao, Mu; Hua, Sheng; Vignarajan, Soma; Jardine, Greg; Hambly, Brett D; Sved, Paul; Dong, Qihan

    2010-04-01

    Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

  4. 2-[2-[4-[2-[2-[ 1,3-Dihydro- 1,1-bis (4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5′-N-ethylcarboxamidoadenosine (FITC-APEC): A Fluorescent Ligand For A2a-Adenosine Receptors

    PubMed Central

    McCabe, R. Tyler; Skolnick, Phil; Jacobson, Kenneth A.

    2011-01-01

    The fluorescein conjugate, FITC-APEC (2-[2-[4-[2-[2-[1,3-dihydro-l,l-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine), is a novel ligand derived from a series of functionalized congeners that act as selective A2a-adenosine receptor agonists. The binding of FITC-APEC to bovine striatal A2a,-adenosine receptors measured by fluorescence techniques was saturable and of a high affinity, with a Bmax, of 2.3 ± 0.3 pmol/mg protein and KD of 57 ± 2 nM. The KD value estimated by fluorescence was consistent with the Ki (11 ± 0.3 nM) obtained by competition studies with [3H]CGS 21680. Additionally, the Bmax, value found by FITC-APEC measurement was in agreement with Bmax, values obtained using radioligand binding. FITC-APEC exhibited rapid and reversible binding to bovine striatum. The potencies of chemically diverse A2a-adenosine receptor ligands estimated by inhibition of FITC-APEC binding were in good agreement with their potencies determined using radioligand binding techniques (r = 0.97, P = 0.0003). FITC-APEC binding was not altered by purine derivatives that do not recognize A2a-adenosine receptors. These findings demonstrate that the novel fluorescent ligand FITC-APEC can be used in the quantitative characterization of ligand binding to A2a-adenosine receptors. PMID:23772170

  5. Aldosterone alters the participation of endothelial factors in noradrenaline vasoconstriction differently in resistance arteries from normotensive and hypertensive rats.

    PubMed

    Xavier, Fabiano E; Blanco-Rivero, Javier; Avendaño, María Soledad; Sastre, Esther; Yela, Rubén; Velázquez, Kyra; Salaíces, Mercedes; Balfagón, Gloria

    2011-03-11

    This study analyzed the effect of aldosterone (0.05mg/kg per day, 3 weeks) on vasoconstriction induced by noradrenaline in mesenteric resistance arteries from WKY rats and SHR. Contraction to noradrenaline was measured in mesenteric resistance arteries from untreated and aldosterone-treatedrats from both strains. Participation of nitric oxide (NO), superoxide anions, thromboxane A(2) (TxA(2)) and prostacyclin in this response was determined. 6-keto-prostaglandin (PG)F1alpha and thromboxane B(2) (TxB(2)) releases were determined by enzyme immunoassay. NO and superoxide anion release were also determined by fluorescence and chemiluminiscence, respectively. Aldosterone did not modify noradrenaline-induced contraction in either strain. In mesenteric resistance arteries from both aldosterone-treated groups, endothelium removal or preincubation with NO synthesis inhibitor L-NAME increased the noradrenaline-induced contraction, while incubation with the superoxide anion scavenger tempol decreased it. Preincubation with either the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively) decreased the noradrenaline contraction in aldosterone-treated animals, while this response was not modified by COX-1 inhibitor SC-560. TxA(2) synthesis inhibitor (furegrelate), or TxA2 receptor antagonist (SQ 29 548) also decreased the noradrenaline contraction in aldosterone-treated animals. In untreated SHR, but not WKY rats, this response was increased by L-NAME, and reduced by tempol, indomethacin, NS-398 or SQ 29 548. Aldosterone treatment did not modify NO or TxB(2) release, but it did increase superoxide anion and 6-keto-PGF(1alpha) release in mesenteric resistance arteries from both strains. In conclusion, chronic aldosterone treatment reduces smooth muscle contraction to alpha-adrenergic stimuli, producing a new balance in the release of endothelium-derived prostanoids and NO.

  6. Blood pressure and vascular reactivity changes in spontaneously hypertensive rats fed fish oil.

    PubMed Central

    Yin, K.; Chu, Z. M.; Beilin, L. J.

    1991-01-01

    1. To examine possible mechanisms of antihypertensive effects of feeding fish oil rich in n-3 fatty acids, we have studied vascular reactivity of aortic rings and perfused mesenteric resistance vessels of spontaneously hypertensive rats (SHR) given such a diet. 2. In two experiments, rats were fed a semi-synthetic diet containing either 'fish oil' (10 and 20% by weight) or hydrogenated coconut oil (control) (10 and 20%) for 4 weeks. 3. Blood pressure rose significantly less in the fish oil group than in controls in both experiments. 4. Aortic rings from control rats showed endothelium-dependent relaxations to low concentrations of acetylcholine (ACh) but relaxed less at higher concentrations. In contrast, rings from the fish oil group had relaxations which increased through the range of concentrations used. Indomethacin (10 microM) also increased the relaxation responses seen in rings from control rats, suggesting that fish oil inhibits a contractile cyclo-oxygenase product. This contractile substance may be thromboxane A2 (TxA2) or its endoperoxide precursor, prostaglandin H2 (PGH2) as aortic incubates and serum levels of TxB2 (the stable product of TxA2) were greatly reduced in fish oil-fed rats, and the decrease of relaxant responses to high concentrations of ACh were also blocked by a TxA2/PGH2 receptor blocker (SQ 29548). 5. In contrast to aortic rings, perfused preconstricted mesenteric resistance vessels of control rats relaxed to ACh in a similar fashion to tissues from fish oil-fed rats. However, in this preparation, fish oil feeding enhanced relaxations to sodium nitroprusside (SNP) and contractile responses to noradrenaline were less than controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1855129

  7. Adenosine A1 receptor: Functional receptor-receptor interactions in the brain

    PubMed Central

    Sichardt, Kathrin

    2007-01-01

    Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A1 receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A2A, A3, neuropeptide Y, and purinergic P2Y1 receptors will be described in the first part. The second part deals with interactions between A1Rs and ionotropic receptors, especially GABAA, NMDA, and P2X receptors as well as ATP-sensitive K+ channels. Finally, the review will discuss new approaches towards treating neurological disorders. PMID:18404442

  8. Cyclooxygenase-2 Inhibition Restored Endothelium-Mediated Relaxation in Old Obese Zucker Rat Mesenteric Arteries

    PubMed Central

    Vessières, Emilie; Belin de Chantemèle, Eric J.; Toutain, Bertrand; Guihot, Anne-Laure; Jardel, Alain; Loufrani, Laurent; Henrion, Daniel

    2010-01-01

    Metabolic syndrome is associated with reduced endothelial vasodilator function. It is also associated with the induction of cyclooxygenase-2 (COX2), which produces vasoactive prostanoids. The frequency of metabolic syndrome increases with age and aging per se is a risk factor associated with reduced endothelium-mediated relaxation. Nevertheless, the combined effect of aging and metabolic syndrome on the endothelium is less known. We hypothesized that COX2 derived prostanoids may affect endothelium function in metabolic syndrome associated with aging. We used obese Zucker rats, a model of metabolic syndrome. First order mesenteric arteries were isolated from 4- and 12-month-old rats and acetylcholine (endothelium)-dependent relaxation determined using wire-myography. Endothelium-mediated relaxation, impaired in young Zucker rats (89 versus 77% maximal relaxation; lean versus Zucker), was further reduced in old Zucker rats (72 versus 51%, lean versus Zucker). The effect of the nitric oxide-synthesis inhibitor L-NAME on the relaxation was reduced in both young and old Zucker rats without change in eNOS expression level. COX inhibition (indomethacin) improved acetylcholine-mediated relaxation in old obese rats only, suggesting involvement of vasoconstrictor prostanoids. In addition, COX2 inhibition (NS398) and TxA2/PGH2 receptor blockade (SQ29548) both improved relaxation in old Zucker rat arteries. Old Zucker rats had the highest TxB2 (TxA2 metabolite) blood level associated with increased COX2 immunostaining. Chronic COX2 blockade (Celecoxib, 3 weeks) restored endothelium-dependent relaxation in old Zucker rats to the level observed in old lean rats. Thus the combination of aging and metabolic syndrome further impairs endothelium-dependent relaxation by inducing an excessive production of COX2-derived vasoconstrictor(s); possibly TxA2. PMID:21423385

  9. Cyclooxygenase-2 inhibition restored endothelium-mediated relaxation in old obese zucker rat mesenteric arteries.

    PubMed

    Vessières, Emilie; Belin de Chantemèle, Eric J; Toutain, Bertrand; Guihot, Anne-Laure; Jardel, Alain; Loufrani, Laurent; Henrion, Daniel

    2010-01-01

    Metabolic syndrome is associated with reduced endothelial vasodilator function. It is also associated with the induction of cyclooxygenase-2 (COX2), which produces vasoactive prostanoids. The frequency of metabolic syndrome increases with age and aging per se is a risk factor associated with reduced endothelium-mediated relaxation. Nevertheless, the combined effect of aging and metabolic syndrome on the endothelium is less known. We hypothesized that COX2 derived prostanoids may affect endothelium function in metabolic syndrome associated with aging. We used obese Zucker rats, a model of metabolic syndrome. First order mesenteric arteries were isolated from 4- and 12-month-old rats and acetylcholine (endothelium)-dependent relaxation determined using wire-myography. Endothelium-mediated relaxation, impaired in young Zucker rats (89 versus 77% maximal relaxation; lean versus Zucker), was further reduced in old Zucker rats (72 versus 51%, lean versus Zucker). The effect of the nitric oxide-synthesis inhibitor L-NAME on the relaxation was reduced in both young and old Zucker rats without change in eNOS expression level. COX inhibition (indomethacin) improved acetylcholine-mediated relaxation in old obese rats only, suggesting involvement of vasoconstrictor prostanoids. In addition, COX2 inhibition (NS398) and TxA2/PGH2 receptor blockade (SQ29548) both improved relaxation in old Zucker rat arteries. Old Zucker rats had the highest TxB2 (TxA2 metabolite) blood level associated with increased COX2 immunostaining. Chronic COX2 blockade (Celecoxib, 3 weeks) restored endothelium-dependent relaxation in old Zucker rats to the level observed in old lean rats. Thus the combination of aging and metabolic syndrome further impairs endothelium-dependent relaxation by inducing an excessive production of COX2-derived vasoconstrictor(s); possibly TxA2.

  10. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    PubMed

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D

    2010-12-29

    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  11. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  12. Regulation of Activator Protein-1 by 8-iso-Prostaglandin E{sub 2} in a Thromboxane A{sub 2} Receptor-Dependent and -Independent Manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP i