Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Anti-inflammatory effect of a novel locally acting A<Subscript>2A</Subscript> receptor agonist in a rat model of oxazolone-induced colitis

Adenosine represents a powerful modulating factor, which has been shown to orchestrate the scope, duration, and remission of the inflammatory response through the activation of four specific receptors, classified as A₁, A_2A, A_2B, and A₃, all being widely expressed in a variety of immune cells. Several selective A_2A receptor agonists have displayed anti-inflammatory effects, through the suppression of IL-12, TNF, and IFN-γ production by monocytes and lymphocytes, in the setting of chronic intestinal inflammation. However, the therapeutic application of A_2A receptor agonists remains hindered by the risk of serious cardiovascular adverse effects arising from the wide systemic distribution of A_2A receptors. The present study focused on evaluating the anti-inflammatory effects of the novel poorly absorbed A_2A receptor agonist PSB-0777 in a rat model of oxazolone-induced colitis as well as to evaluate its cardiovascular adverse effects, paying particular attention to the onset of hypotension, one of the main adverse effects associated with the systemic pharmacological activation of A_2A receptors. Colitis was associated with decreased body weight, an enhanced microscopic damage score and increased levels of colonic myeloperoxidase (MPO). PSB-0777, but not dexamethasone, improved body weight. PSB-0777 and dexamethasone ameliorated microscopic indexes of inflammation and reduced MPO levels. The beneficial effects of PSB-0777 on inflammatory parameters were prevented by the pharmacological blockade of A_2A receptors. No adverse cardiovascular events were observed upon PSB-0777 administration. The novel A_2A receptor agonist PSB-0777 could represent the base for the development of innovative pharmacological entities able to act in an event-specific and site-specific manner.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A<Subscript>2B</Subscript> adenosine receptors

Adenosine A_2B receptors (A_2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A_2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A_2BR localization was examined by immunofluorescence. The role of A_2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A_2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A_2BR antagonist MRS1754 enhanced electrically induced NK₁-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A_2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A_2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A_2BR expression. A_2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.     

Tritium-labeled agonists as tools for studying adenosine A<Subscript>2B</Subscript> receptors

A selective agonist radioligand for A_2B adenosine receptors (A_2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A_2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [³H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A_2BAR agonist [³H]NECA for its potential to label A_2BARs. [³H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A_2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [³H]NECA than versus the A_2B-antagonist radioligand [³H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A_2BAR agonist, it displayed higher affinity versus [³H]PSB-603 than versus [³H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A_2BAR. In conclusion, [³H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A_2BAR conformation.     

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A_2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A₁, A_2A, and A_2B ARs. Cell proliferation was measured by [³H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A₁), BAY60-6583 (A_2B), and IB-MECA (A₃), and the antagonists PSB-36 (A₁), MSX-2 (A_2A), and PSB-10 (A₃) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A_2A), and the antagonists preladenant (SCH-420814, A_2A), PSB-1115 (A_2B), and PSB-603 (A_2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC₅₀ value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.     

Adenosine A<Subscript>2A</Subscript> receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies

We aim to investigate whether A_2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A_2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A_2A agonist, 24 h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC₅₀ 10^?9.2 M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (～1.5-fold) and pre-eclamptic pregnancies (～1.2-fold), an effect blocked by the A_2A antagonist, ZM-241385 (10^?5 M) and the inhibitor of NO synthase, N _ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A_2A. CGS-21680 also increased (～1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A_2A decrease (～30 %) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A_2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.     

Modulation of protein kinase C by adenosine: Involvement of adenosine A1 receptor-pertussis toxin sensitive nucleotide binding protein system

The objective of this study was to determine whether adenosine A₁ or A₂ receptor was responsible for the regulation of protein kinase C (PKC) in porcine coronary artery and its coupling to G-protein. Endothelium denuded arterial rings were incubated with PDBu (200nM) in the presence or absence of adenosine receptor agonists and antagonists for 1 day. Following incubation, the arterial rings were contracted with increasing concentrations of endothelin-1 (ET-1) (10^–10–10^–7M). Arteries incubated with PDBu alone failed to produce contraction in response to ET-1. On the contrary, inclusion of A₁ receptor agonist ENBA at 10^–9M in the incubation media with PDBu protected against the PDBu induced blunting of the ET-1 contractions by 50%. Incubation with ENBA alone increased ET-1 dependent contractions by about 2 fold. Inclusion of A₁ receptor antagonist, N0861 at 10^–6 M along with PDBu and ENBA, completely blocked the protective effect of ENBA against the PDBu induced attenuation of ET-1 contractions. N0861 also completely blocked the increase in ET-1 contractions in the arterial rings incubated with ENBA alone. Another A₁ receptor antagonist DPCPX also produced similar results as N0861. On the contrary, arterial rings incubated with relatively specific A₂ receptor agonist CGS 21680 at 10^–4M did not produce any protection against PDBu induced blunting of the ET-1 contractions. Incubation with CGS 21680 alone also did not significantly alter the ET-1 contractions. Interestingly, inclusion of A₂ receptor antagonist DMPX at 10^–4M in the incubation media along with CGS 21680 mimicked the effects of ENBA alone i.e. produced protection against PDBu and enhanced ET-1 contractions. Incubation of the arteries with ENBA alone caused an accumulation of PKC levels, whereas, incubation with CGS 21680 had no significant effect on PKC levels. To study the coupling of adenosine receptor with G-protein, the tissue was incubated for one day with cholera (CT) or pertussis toxin (PT) in the presence or absence or ENBA and PDBu as described above. Incubation with PT blocked the protective effect of ENBA against PDBu as well as the elevation of ET-1 response when incubated with ENBA alone. On the contrary, incubation with CT did not produce any significant effect on ENBA responses. These results indicate that PKC is modulated by adenosine via A₁ adenosine receptors and through a PT sensitive G-protein.This work was supported by National Heart, Lung and Blood Institute Grant HL-27339.     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A2A receptors and endogenous adenosine

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N⁶‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A_2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A_2A receptors. The A_2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A_2A receptors modulators.     

Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson’s disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

Inhibition of inflammation-induced alterations in rat small intestine by the herbal preparations STW 5 and STW 6

Inflammation is a common mechanism of many gastrointestinal diseases. Therefore, it is interesting to know, whether complex phytopharmaceuticals known to modulate gastrointestinal motor function reveal also anti-inflammatory properties. We tested the fixed herbal combination product STW 5 (Iberogast^®) and its main component Iberis amara fresh plant extract (STW 6) to characterize their protective potential in an experimental inflammation model in vitro. The test system consisted of ileum/jejunum segments from male Wistar rats. Inflammation was evoked by intraluminal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) for 30 min. Preincubation of TNBS together with STW 5 and STW 6 prevented the TNBS-induced inhibition of ACh-induced contractions. No differences were found between water-dissolved and ethanol-dissolved extracts. STW 5 and STW 6 reduced morphological changes induced by TNBS in mucosal and muscle layers. The IL-10 mRNA measured by qRT-PCR was not influenced by TNBS but increased by STW 5 and STW 6. The TNBS-induced increase in the TNFα-mRNA expression was suppressed by STW 5 but not by STW 6. Additionally, STW 5 decreased TNFα release in LPS-stimulated human monocytes. STW 6 influenced neither the TNFα-mRNA nor the TNFα release. These findings demonstrate that STW 5 reduced inflammation-induced alterations in ileum/jejunum segments. The effects were associated with a restoration of the disturbed ACh-induced contraction, pathohistological protection and inhibition of TNFα. STW 6 may contribute to the protective effect of STW 5 mainly by increasing IL-10 pathway but not by influencing TNFα.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Enhanced 5-HT<Subscript>2A</Subscript> Receptors in Brain Stem and ALDH Activity in Brain Stem and Liver: 5-HT<Subscript>2A</Subscript> Regulation on ALDH in Primary Hepatocytes Cultures In Vitro

Brain serotonin (5-HT) modulates the neural effects of ethanol. In the present study, we investigated the changes in 5-HT level, 5-HT_2A receptor binding and aldehyde dehydrogenase (ALDH) activity in brain stem and liver of ethanol treated rats and 5-HT_2A regulation on ALDH in hepatocyte cultures in vitro. The 5-HT content in the brain stem and liver significantly decreased with an increased 5-HIAA/5-HT ratio in the ethanol treated rats compared to control. Scatchard analysis of [³H] (±)2,3-dimethoxyphenyl-1-[2-(-4-piperidine)-methanol] [³H] MDL 100907 against ketanserin in brain stem of ethanol treated rats showed a significant increase in B _max without any change in K _d compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in both control and ethanol treated rats with unity as Hill slope value. A significant increase in V _max of ALDH activity in liver and a significant decrease in K _m in liver and brain stem of ethanol treated rats compared to control was observed. In 24 h culture studies, an increase in enzyme activity was observed in cells in medium with 10% ethanol. The elevated ALDH activity in ethanol treated cells was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT. Ketanserin, an antagonist of 5-HT_2A, reversed the effect of 5HT on 10% ethanol induced ALDH activity in hepatocytes. Our results showed that there was a decreased 5-HT content with an enhanced 5-HT_2A receptor and aldehyde dehydrogenase activity in the brain stem of alcohol treated rats and in vitro hepatocyte cultures. The enhanced ALDH activity in ethanol supplemented hepatocytes was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT.     

Molecular modeling of a PAMAM-CGS21680 dendrimer bound to an A2A adenosine receptor homodimer

The theoretical possibility of bivalent binding of a dendrimer, covalently appended with multiple copies of a small ligand, to a homodimer of a G protein-coupled receptor was investigated with a molecular modeling approach. A molecular model was constructed of a third generation (G3) poly(amidoamine) (PAMAM) dendrimer condensed with multiple copies of the potent A_2A adenosine receptor agonist CGS21680. The dendrimer was bound to an A_2A adenosine receptor homodimer. Two units of the nucleoside CGS21680 could occupy the A_2A receptor homodimer simultaneously. The binding mode of CGS21680 moieties linked to the PAMAM dendrimer and docked to the A_2A receptor was found to be similar to the binding mode of a monomeric CGS21680 ligand.     

Caffeine and a selective adenosine A<Subscript>2A</Subscript> receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

Background  

Caffeine, a nonselective adenosine A₁ and A_2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A_2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinson's disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A_2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum.     

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A_2A receptor (A_2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A_2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A_2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A_2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A_2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A_2AR-mediated Col1 increase. In contrast, A_2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A_2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A_2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.