Adenosine A1 and A2A receptor regulation of protein phosphatase 2A in the murine heart

Adenosine plays a role in regulating the contractile function of the heart. This includes a positive ionotropic action via the adenosine A(2A) receptor (A(2A)R) and an inhibition of beta(1)-adrenergic receptor-induced ionotropy (antiadrenergic action) via the adenosine A(1) receptor (A(1)R). Phosphatase activity has also been shown to influence contractile function by affecting the level of protein phosphorylation. Protein phosphatase 2A (PP2A) plays a significant role in mediating the A(1)R antiadrenergic effect. The purpose of this study was to investigate the effects of A(2A)R and A(1)R on the activities of PP2A in hearts obtained from wild-type (WT) and A(2A)R knockout (A(2A)R-KO) mice. PP2A activities were examined in myocardial particulate and cytoplasmic extract fractions. Treatment of wild-type hearts with the A(1)R agonist CCPA increased the total PP2A activity and increased the particulate:cytoplasmic PP2A activity ratio. Treatment with the A(2A)R agonist CGS-21680 (CGS) decreased the total PP2A activity and decreased the particulate:cytoplasmic PP2A activity ratio. This indicated a movement of PP2A activity between cell fractions. The effect of CCPA was inhibited by CGS. In A(2A)R-KO hearts the response to A(1)R activation was markedly enhanced whereas the response to A(2A)R activation was absent. These data show that A(2A)R and A(1)R regulate PP2A activity, thus suggesting an important mechanism for modulating myocardial contractility.     

Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury

Inactivation of the adenosine A(2A) receptor (A(2A)R) consistently protects against ischemic brain injury and other neural insults, but the relative contribution of A(2A)Rs on peripheral inflammatory cells versus A(2A)Rs expressed on neurons and glia is unknown. We created a chimeric mouse model in which A(2A)Rs on bone marrow-derived cells (BMDCs) were selectively inactivated or reconstituted by bone marrow transplantation. Selective reconstitution of A(2A)Rs on BMDCs (A(2A)R knockout mice transplanted with wild-type bone marrow cells) largely reinstates ischemic brain injury in global A(2A)R knockout mice. Conversely, selective inactivation of A(2A)Rs on BMDCs (wild-type mice transplanted with A(2A)R knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain, but exacerbates ischemic liver injury. These results indicate that the A(2A)R-stimulated cascade in BMDCs is an important modulator of ischemic brain injury and that ischemic brain and liver injuries are regulated distinctly by A(2A)Rs on BMDCs.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.     

On the existence of a possible A2A-D2-β-Arrestin2 complex: A2A agonist modulation of D2 agonist-induced β-arrestin2 recruitment

Given that coactivation of adenosine A(2A) (A(2A)R) and dopamine D(2) (D(2)R) receptors results in the coaggregation, cointernalization, and codesensitization of the A(2A)R and D(2)R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A(2A)R agonist CGS21680 in A(2A)R-D(2)R-coexpressing cells to modulate the D(2)R agonist-induced recruitment of β-arrestin2 to the D(2)R by means of proximity-based bioluminescence resonance energy transfer (BRET(2)) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET(2) signal between β-arrestin2(RLuc) and D(2L)R(GFP2) upon D(2)R activation, by increasing the potency of the D(2)R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2(GFP2) and D(2L)R(YFP) as seen from the co-trafficking analysis. Furthermore, the A(2A)R agonist advanced the time for the increase in Akt phosphorylation obtained with the D(2)R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A(2A)R-D(2)R heteromers. Taken together, the results indicate that the antagonistic A(2A)R-D(2)R allosteric receptor-receptor interaction in A(2A)R-D(2)R heteromers favors β-arrestin2 recruitment to the D(2L)R protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.     

Protein phosphatase 2A inhibitors, I(1)(PP2A) and I(2)(PP2A), associate with and modify the substrate specificity of protein phosphatase 1

Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A.     

Role of CYP epoxygenases in A2A AR-mediated relaxation using A2A AR-null and wild-type mice

We hypothesized that A2A adenosine receptor (A2A AR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2A AR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10(-6) M), an adenosine analog, caused relaxation in wild-type A2A AR (A2A AR+/+; +33.99 +/- 4.70%, P < 0.05) versus contraction in A2A AR knockout (A2A AR(-/-); -27.52 +/- 4.11%) mouse aortae. An A2A AR-specific antagonist (SCH-58261; 1 microM) changed the NECA (10(-6) M) relaxation response to contraction (-35.82 +/- 4.69%, P < 0.05) in A2A AR+/+ aortae, whereas no effect was noted in A2A AR(-/-) aortae. Significant contraction was seen in the absence of the endothelium in A2A AR+/+ (-2.58 +/- 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester; 100 microM) and a cyclooxygenase inhibitor (indomethacin; 10 microM) failed to block NECA-induced relaxation in A2A AR+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 microM) changed NECA-mediated relaxation (-22.74 +/- 5.11% at 10(-6) M) and CGS-21680-mediated relaxation (-18.54 +/- 6.06% at 10(-6) M) to contraction in A2A AR+/+ aortae, whereas no response was noted in A2A AR(-/-) aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 microM] was able to block NECA-induced relaxation in A2A AR+/+ aortae, whereas omega-hydroxylase inhibitors (10 microM dibromo-dodecenyl-methylsulfimide and 10 microM HET-0016) changed contraction into relaxation in A2A AR(-/-) aorta. Cyp2c29 protein was upregulated in A2A AR+/+ aortae, whereas Cyp4a was upregulated in A2A AR(-/-) aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2A AR+/+ versus A2A AR(-/-) aortae. EET levels were not significantly different between A2A AR+/+ and A2A AR(-/-) aortae. It is concluded that CYP epoxygenases play an important role in A2A AR-mediated relaxation, and the deletion of the A2A AR leads to contraction through Cyp4a.     

Functional expression of single-chain heterodimeric G-protein-coupled receptors for adenosine and dopamine

The direct homo- and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A(2A)R) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A(2A)R/A(2A)R vs. A(2A)R/D2R). In order to stimulate the heteromerization of A(2A)R and D2R, we have designed a single-polypeptide-chain heterodimeric A(2A)R/D2R complex by fusing the C-terminus of the A(2A)R via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A(2A)R/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.     

Novel 1,3-dipropyl-8-(1-heteroarylmethyl-1H-pyrazol-4-yl)-xanthine derivatives as high affinity and selective A2B adenosine receptor antagonists

A series of new 1,3-dipropyl-8-(1-heteroarylmethyl-1H-pyrazol-4-yl)-xanthine derivatives as A(2B)-AdoR antagonists have been synthesized and evaluated for their binding affinities for the A(2B), A(1), A(2A), and A(3)-AdoRs. 8-(1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazol-4-yl)-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione (4) displayed high affinity (K(i)=1 nM) and selectivity for the A(2B)-AdoR versus A(1), A(2A), and A(3)-AdoRs (A(1)/A(2B), A(2A)/A(2B), and A(3)/A(2B) selectivity ratios of 370, 1100, and 480, respectively). The synthesis and SAR of this novel class of compounds are presented herein.     

Insilico study of the A2AR–D2R kinetics and interfacial contact surface for heteromerization

G-protein-coupled receptors (GPCRs) are cell surface receptors. The dynamic property of receptor-receptor interactions in GPCRs modulates the kinetics of G-protein signaling and stability. In the present work, the structural and dynamic study of A(2A)R-D(2)R interactions was carried to acquire the understanding of the A(2A)R-D(2)R receptor activation and deactivation process, facilitating the design of novel drugs and therapeutic target for Parkinson's disease. The structure-based features (Alpha, Beta, SurfAlpha, and SurfBeta; GapIndex, Leakiness and Gap Volume) and slow mode model (ENM) facilitated the prediction of kinetics (K (off), K (on), and K (d)) of A(2A)R-D(2)R interactions. The results demonstrated the correlation coefficient 0.294 for K (d) and K (on) and the correlation coefficient 0.635 for K (d) and K (off), and indicated stable interfacial contacts in the formation of heterodimer. The coulombic interaction involving the C-terminal tails of the A(2A)R and intracellular loops (ICLs) of D(2)R led to the formation of interfacial contacts between A(2A)R-D(2)R. The properties of structural dynamics, ENM and KFC server-based hot-spot analysis illustrated the stoichiometry of A(2A)R-D(2)R contact interfaces as dimer. The propensity of amino acid residues involved in A(2A)R-D(2)R interaction revealed the presence of positively (R, H and K) and negatively (E and D) charged structural motif of TMs and ICL3 of A(2A)R and D(2)R at interface of dimer contact. Essentially, in silico structural and dynamic study of A(2A)R-D(2)R interactions will provide the basic understanding of the A(2A)R-D(2)R interfacial contact surface for activation and deactivation processes, and could be used as constructive model to recognize the protein-protein interactions in receptor assimilations.     

In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis

In rat hepatocytes, the role of cAMP and Ca(2+) as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A(1), A(2A), A(2B) and A(3). Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A(1) and A(3) adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)). Accordingly, the A(1) or A(3)-mediated increase in [Ca(2+)](cyt) and in ureagenic activity were both inhibited by chelating Ca(2+) with either EGTA or BAPTA-AM. Also, Gd(3+) blocked both the increase in [Ca(2+)](cyt) and ureagenesis, suggesting that a Ca(2+) channel may be involved in the response to both A(1) and A(3). A partial effect was observed with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A(2A) or the A(2B) adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A(1) or A(3). The stimulation of either the A(2A) or A(2B) adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd(3+) or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A(2A) and A(2B), but not the response to either A(1) or A(3). Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A(1) and A(3) adenosine receptor subtypes is [Ca(2+)](cyt), while the message from the A(2A) and A(2B) adenosine receptor subtypes is relayed by [cAMP].     

Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors and dopamine D2 receptors

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A(2A) receptors (A(2A)R) and dopamine D(2) receptors (D(2)R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D(2)R and in cultured striatal cells. A(2A)R/D(2)R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D(2)R-transfected SH-SY5Y cells and from mouse fibroblast Ltk(-) cells stably transfected with human D(2)R (long form) and transiently cotransfected with the A(2A)R double-tagged with hemagglutinin. Long term exposure to A(2A)R and D(2)R agonists in D(2)R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A(2A)R and D(2)R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D(2)R are involved.     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.     

Studies on the effect of diadenlyated nucleotides on calcium mobilization and prostacyclin synthesis in bovine aortic endothelial cells

Extracellular adenine dinucleotides are modulators of blood vessel tone. We have previously demonstrated that Ap(2)A and Ap(4)A induce the synthesis of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) while Ap(3)A and Ap(5)A do not [FEBS Lett. 427 (1998) 320; Arch. Biochem. Biophys. 364 (1999) 280.]. In this communication we determine the effect of Ap(x)As (x=2-5) on prostacyclin (PGI(2)) synthesis and Ca(2+) mobilization in BAEC. Ap(2)A and Ap(4)A significantly enhanced the synthesis of PGI(2) while Ap(3)A and Ap(5)A do not. These data support the notion that Ap(2)A and Ap(4)A are vasodilators. All four dinucleotides significantly enhanced Ca(2+) mobilization over basal levels. Ap(5)A and Ap(3)A enhanced 2.0 and 1.6 times more Ca(2+) release than Ap(4)A, respectively. Since neither Ap(5)A nor Ap(3)A enhanced the synthesis of either PGI(2) or NO but did mobilize Ca(2+), these data support the hypothesis that in BAEC Ca(2+) release is localized or compartmentalized.     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.     

Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists

Striatal adenosine A(2A) receptors (A(2A)Rs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D(2) receptors (D(2)Rs). A(2A)Rs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1) receptors (A(1)Rs). It has been hypothesized that postsynaptic A(2A)R antagonists should be useful in Parkinson's disease, while presynaptic A(2A)R antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2A)R antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2A)R-D(2)R and A(1)R-A(2A)R heteromers to determine possible differences in the affinity of these compounds for different A(2A)R heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2A)R when co-expressed with D(2)R than with A(1)R. KW-6002 showed the best relative affinity for A(2A)R co-expressed with D(2)R than co-expressed with A(1)R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

1,3-Dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives as highly potent and selective human A(2B) adenosine receptor antagonists

A new series of 1,3-dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives has been identified as potent A(2B) adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A(2B), A(1), A(2A), and A(3) adenosine receptors. N-(4-chloro-phenyl)-2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl] (11c) showed a high affinity for the human A(2B) adenosine receptor K(i)=7nM and good selectivity (A(1), A(2A), A(3)/A(2B)>140). Synthesis and SAR of this novel class of compounds is presented herein.