Modulating effect of adenosine deaminase on function of adenosine A1 receptors

AIM: To study the modulating effect of adenosine deaminase (ADA) on the adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R. METHODS: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay. RESULTS: HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that the K(D) value and B(max) value of [3H]DPCPX were 1.6+/-0.2 nmol/L and 1.819+/-0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3+/-0.2 nmol/L and 1.992+/-0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K(D) value and B(max) value of [3H]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist CCPA/[3H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K(H) value of 0.74 (0.11+/-4.8) nmol/L (intact cells) or 1.8 (0.25+/-10) nmol/L (membrane) and a K(L) value of 0.94 (0.62+/-1.41) micromol/L (intact cells) or 0.77 (0.29+/-0.99) micromol/L (membrane). The K(L) value is not significantly different from the IC50 value of 0.84(0.57+/-1.23) micromol/L (intact cells) or 0.84 (0.63+/-1.12) micromol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[3H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC50 value of CPA were 10 (5+/-29) nmol/L and 94 (38+/-229) nmol/L in the presence or absence of ecto-ADA, respectively. CONCLUSION: A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells.     

Agonist photoaffinity labeling of A1 adenosine receptors: persistent activation reveals spare receptors

This study describes experiments investigating the mechanism of activation of A1 adenosine receptors. Isolated rat fat cells were used as a cellular model. The A1 receptors of these cells were covalently labeled with the agonist photoaffinity label R-2-azido-N6-p-hydroxyphenylisopropyladenosine. The covalent incorporation of the label into the binding subunit of the receptor was verified by demonstration of specific labeling of a peptide with Mr = 35,000 by the radioiodinated label. Such covalent labeling followed by removal of label not covalently bound led to a concentration-dependent reduction of cellular cAMP levels. This persistent effect of covalent labeling occurred with an IC50 value of 9 nM compared to an IC50 value of 0.9 nM for the direct reduction of cAMP levels by the label. The affinity of the label was determined in binding experiments. The Ki value of 19 nM was about 20 times higher than the corresponding IC50 value of cAMP reduction. Finally, the comparison between covalent binding and its effects suggests that covalently labeled receptors were fully activated. The data are interpreted as evidence for a receptor activation according to the occupancy theory. The analysis of the various concentration-response curves reveals the presence of spare receptors, which can be demonstrated by the method of agonist photoaffinity labeling.     

Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 microM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2'-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5'-amino-5'-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.     

Potent adenosine A1 and A2A receptors antagonists: recent developments

This review summarizes the current tendencies observed in the past 5 years in the development of A(1) and A(2A) adenosine receptor antagonists performed in various academia and industry. A(1) and A(2A) AR antagonists are as well xanthines as heteroaromatic derivatives and are most commonly 6:5 fused heteroatomic compounds. Among xanthine-based compounds, some common features could be pointed out. The recent A(1) AR ligands which show good biological profile, possess long alkyl chains in position 1 and 3 as well as bulky C(8)-substituent, while A(2A) AR antagonists with a high A(2A) AR affinity are C(8)-styryl substituted with N(1)-alkyl/alkynyl moiety or fused tricyclic xanthines possessing heteroatom(s) in the third cycle. The research in the field of heteroaromatic A(1) and A(2A) ARs antagonists impressively has a wide range. Ligands are as well non-fused monocyclic substituted compounds as fused bi- and tricyclic derivatives with the nitrogen, oxygen and sulfur heteroatoms. Most often, adenosine A(1) receptor non-xanthine antagonists are adenine-based, having substituted amino group and variable nitrogen atoms positions in the molecules. A(2A) AR ligands show good affinity when furanyl function, which is crucial for binding, is present in the fused bicyclic and tricyclic analogs. Moreover, tricyclic nitrogen containing antagonists in order to be active, frequently possess long-alkylphenyl moiety.     

Analysis of agonist-antagonist interactions at A1 adenosine receptors

Previous work from our laboratory using sucrose gradient centrifugation and the antagonist radioligand [3H]xanthine amine congener led us to propose that A1 adenosine receptors are coupled to a GTP-binding protein (G protein) in the absence of an agonist and that adenosine receptor antagonists bind to free uncoupled receptors with high affinity and coupled receptors with low affinity and cause a destabilization of receptor-G protein complexes [Mol. Pharmacol. 36:412-419 (1989)]. Because agonists form high affinity ternary complexes composed of the agonist, receptor, and G protein, this hypothesis would imply that interactions between adenosine receptor agonists and antagonists, while competitive, would appear to be "noncompetitive" in nature. Interactions between unlabeled and radiolabeled A1 receptor agonist and antagonist ligands have been investigated using bovine cerebral cortical membranes to further probe this point. The availability of both 3H- and 125I-radioligands allowed us to use both single- and dual-isotope experimental designs. Radioligand antagonist-agonist competition curves along with saturation analyses using filtration and centrifugation to isolate bound radioligand suggested that agonists bind to two sites or receptor states with high affinity and to one site with low affinity. Agonist radioligand saturation curves with or without unlabeled antagonist suggested that antagonists do not bind to all states of the receptor with equal affinity. The computer program EQUIL was used to define models capable of simultaneously fitting all parts of complex experiments in which 125I-N6-aminobenzyladenosine saturation isotherms with or without 8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and a saturation isotherm of [3H]CPX were performed. The data were not compatible with two-independent site models or with ternary complex models involving one receptor and one G protein. The data were fit by a model involving one receptor and two G proteins and by a model involving two receptors and one G protein. Both models suggest that 1) a high percentage of the receptor(s) is coupled to a G protein in the absence of an agonist and 2) agonists stabilize whereas antagonists destabilize precoupled receptor-G protein complexes. Because of this, competitive interactions between A1 agonists and antagonists appear noncompetitive in nature.     

Predominant role of A1 adenosine receptors in mediating adenosine induced vasodilatation of rat diaphragmatic arterioles: involvement of nitric oxide and the ATP-dependent K+ channels

1. We investigated, by intravital microscopy in rats, the role of the subtypes of adenosine receptors A₁ (A₁/AR) and A₂ (A₂AR) in mediating adenosine-induced vasodilatation of second and third order arterioles of the diaphragm.
2. Adenosine, and the A₁AR selective agonists R(−)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) and N⁶-cyclo-pentyl-adenosine (CPA) induced a similar concentration-dependent dilatation of diaphragmatic arterioles. The non selective A₂AR subtype agonist N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl) ethyl]adenosine (DPMA) also dilated diaphragmatic arterioles but induced a significantly smaller dilatation than adenosine. By contrast the selective A_2aAR subtype agonist 2-[p-(2-carboxyethyl)phenyl amino]-5′-N-ethyl carboxamido adenosine (CGS 21680) did not modify diaphragmatic arteriolar diameter.
3. The non selective adenosine receptor antagonist 1,3-dipropyl-8-p-sulphophenylxanthine (SPX, 100 μM) and the selective A₁AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nM) significantly attenuated adenosine-induced dilatation of diaphragmatic arterioles. By contrast, adenosine significantly dilated diaphragmatic arterioles in the presence of A₂AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM).
4. The dilatation induced by adenosine was unchanged by the mast cell stabilizing agent sodium cromoglycate (cromolyn, 10 μM).
5. The nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine (L-NOARG, 300 μM) attenuated the dilatation induced by adenosine, and by the A₁AR and A₂AR agonists.
6. The ATP-dependent K⁺ channel blocker glibenclamide (3 μM) significantly attenuated diaphragmatic arteriolar dilatation induced by adenosine and by the A₁AR agonists R-PIA and CPA. By contrast, glibenclamide did not significantly modify arteriolar dilatation induced by the A₂AR agonist DPMA.
7. These findings suggest that adenosine-induced dilatation of diaphragmatic arterioles in the rat is predominantly mediated by the A₁AR, via the release of NO and activation of the ATP-dependent K⁺ channels.

     

Effects of enprofylline on A1 and A2 adenosine receptors

The effects of enprofylline were studied on A₁ adenosine receptors of rat fat cells and on A₂ adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5′-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a K_B of 130 μM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A₂ receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N⁶-R-(−)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a K_B of 32 μM. The K_i value for inhibition of [³H]PIA binding to fat cell membranes was 45 μM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with K_i values of 15, 130 and 110 μM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A₁ and A₂ adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline.     

Molecular characterization of A1 and A2a adenosine receptors

Detailed amino acid sequence analyses of A₁ and A_2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A_2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A₁ and A_2a receptors have been introduced. For example, an isothiocyanate, ρ-DITC-APEC (100 nM), irreversibly diminished the B_max for [³H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5′-N-ethylcarboxamido-adenosine) binding in rabbit striatal membranes by 71% (K_d unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A_2a receptors were characterized using [³H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), respectively. The inhibition of A₂ radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A₁ receptors in which either His²⁵¹ or His²⁷⁸ residues have been substituted with Leu suggest that both histidines are important in binding. © 1993 Wiley-Liss, Inc.     

Intrinsic activity at adenosine A1 receptors: partial and inverse agonism

Novel phenomena, such as constitutive activity and inverse agonism, have led to a ligand (re)classification along an agonists-neutral antagonist-inverse agonist continuum. This review focuses on adenosine A(1) receptor ligands and their intrinsic activity (alpha). The intrinsic activity of a ligand depends both on the chemical characteristics of the compound itself and on the experimental conditions in which it is assayed. Consequently, due to this tissue-dependency determination of a ligand s intrinsic activity is not always easily performed. Meanwhile, this feature may also be used in a profitable manner, namely to separate desired and unwanted effects of the drug. Briefly, this review provides possible screening methods to distinguish the different classes of ligands. It also deals with the structural elements and functional groups in the adenosine A(1) receptor ligands that determine their intrinsic activity.     

A1 adenosine receptors and their ligands: overview and recent developments

Potent adenosine receptor (AR) agonists and antagonists with high selectivity for the A1AR subtype have been developed during the past decades. However, some of the compounds considered to be selective may not be as selective in humans as in rats, and may not be very selective versus the new AR subtypes A3 or A2B. Partial agonists have been developed that may exhibit fewer side effects than full agonists. Low water solubility of many A1 antagonists remains a problem. A1 AR antagonists can be classified as neutral antagonists or inverse agonists; the pharmacological consequences of inverse agonism versus neutral antagonism will have to be the subject of future investigations. Some medicinal plants (e.g. Hypericum perforatum and Valeriana officinalis) contain compounds that are antagonists or partial agonists at A1 ARs; effects on ARs may contribute to their pharmacological activity. 18F- and 11C-labeled A1 AR antagonists have been developed for positron emission tomography studies.     

Adenine nucleotide-induced activation of adenosine A(2B) receptors expressed in Xenopus laevis oocytes: involvement of a rapid and localized adenosine formation by ectonucleotidases

We recently demonstrated that extracellular ATP effectively activates adenosine (Ade) A(2B) receptors indirectly through a localized rapid conversion to Ade by ectonucleotidases on the membrane surface of C6Bu-1 rat glioma cells. These responses were observed even in the presence of adenosine deaminase (ADA). Here, we demonstrate that such responses indeed occur in A(2B) receptor-expressing Xenopus laevis oocytes, which possess endogenous ectonucleotidase activity. In oocytes coexpressing the A(2B) receptor and cystic fibrosis transmembrane conductance regulator (CFTR), Ade induced a concentration-dependent increase in a cyclic AMP-activated CFTR current, a response that was inhibited by the P1 antagonist xanthine-amine congener (XAC). A brief application of ATP and beta,gamma-methylene ATP (beta,gamma-MeATP) also induced the CFTR current in a manner similar to that seen with Ade. Among several nucleotide agonists, ADP, AMP, and adenosine-5'-O-(3-thio)triphosphate induced the CFTR current. Although adenine nucleotide-induced CFTR currents were inhibited by XAC, they were highly resistant to ADA treatment; 5 U/ml ADA was required for inhibition of adenine nucleotide-induced CFTR current, whereas 1 U/ml ADA was sufficient to abolish the Ade-induced response. In addition, the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP markedly inhibited the beta,gamma-MeATP-induced response but not the Ade-induced one. These results support our hypothesis that adenine nucleotides are rapidly and locally converted into Ade on the membrane surface, resulting in the activation of A(2B) receptors.     

腺苷A1受体的作用研究进展

局部组织的缺血/缺氧常使细胞外腺苷浓度升高,腺苷通过细胞膜表面的受体将低氧的信号传入细胞内,使细胞启动低氧应对机制。腺苷受体的4种亚型在这个过程中发挥了不同的生理作用,该文是关于A1受体在保护细胞、促进免疫、调节血压及血糖等方面作用的综述。     

Spinal serotonin 5-HT7 and adenosine A1 receptors,as well as peripheral adenosine A1 receptors,are involved in antinociception by systemically administered amitriptyline

The present study explored a link between spinal 5-HT₇ and adenosine A₁ receptors in antinociception by systemic amitriptyline in normal and adenosine A₁ receptor knock-out mice using the 2% formalin test. In normal mice, antinociception by systemic amitriptyline 3 mg/kg was blocked by intrathecal administration of the selective adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 10 nmol. Blockade was also seen in adenosine A₁ receptor +/+ mice, but not in ?/? mice lacking these receptors. In both normal and adenosine A₁ receptor +/+ mice, the selective 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB269970) 3 μg blocked antinociception by systemic amitriptyline, but it did not prevent antinociception in adenosine A₁ receptor ?/? mice. In normal mice, flinching was unaltered when the selective 5-HT₇ receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin (AS-19) 20 μg was administered alone, but increased when co-administered intrathecally with DPCPX 10 nmol or SB269970 3 μg. Intrathecal AS-19 decreased flinching in adenosine A₁ receptor +/+ mice compared to ?/? mice. Systemic amitriptyline appears to reduce nociception by activating spinal adenosine A₁ receptors secondarily to 5-HT₇ receptors. Spinal actions constitute only one aspect of antinociception by amitriptyline, as intraplantar DPCPX 10 nmol blocked antinociception by systemic amitriptyline in normal and adenosine A₁ receptor +/+, but not ?/? mice. Adenosine A₁ receptor interactions are worthy of attention, as chronic oral caffeine (0.1, 0.3 g/L, doses considered relevant to human intake levels) blocked antinociception by systemic amitriptyline in normal mice. In conclusion, adenosine A₁ receptors contribute to antinociception by systemic amitriptyline in both spinal and peripheral compartments.     

A1 adenosine receptors and muscarinic cholinoceptors in myocardial ischemia

The regulation of cardiac A₁ adenosine receptors and M₂ muscarinic cholinoceptors was investigated in ischemic rat hearts. Ischemia was induced in isolated, perfused hearts either by stop (stop-flow) or by reduction (low-flow) of perfusion flow. Receptor densities and affinities were determined by radioligand binding. The mRNA concentrations of the receptors and of control messages were measured by quantitative polymerase chain reactions (PCR). Second messenger coupling of the receptors was evaluated by measuring their inhibition of adenylate cyclase activity.Up to 60 min of stop-flow ischemia and 6 h of lowflow ischemia, cardiac A₁ adenosine receptor density and affinity, and adenosine receptor-mediated inhibition of adenylate cyclase, did not change significantly, compared to non-ischemic hearts. Receptor down-regulation, however, could be induced by perfusion with the A₁ receptor agonist R-phenyl-isopropyl-adenosine (R-PIA) during normal flow. After 6 h of perfusion with R-PIA (0.1 mol/l), A₁ adenosine receptor density was reduced. Agonist-induced receptor down-regulation was not found after perfusion with R-PIA in low-flow ischemia. The density and the affinity of muscarinic cholinoceptors were not affected during stop-flow ischemia up to 1 h either, whereas the density was down-regulated to 75% of controls (P<0.05) after 6 h of low-flow ischemia. This intervention also reduced inhibition of adenylate cyclase via muscarinic cholinoceptors. In non-ischemic hearts, perfusion with carbachol (10 µmol/l) suppressed receptor densities to 72% of control values.No significant changes in the concentration of A₁ adenosine receptor or M₂ cholinoceptor mRNAs occurred during normal flow, stop-flow and low-flow ischemia. Likewise, agonist stimulation with R-PIA or carbachol during normal flow did not change the respective receptor mRNA concentrations significantly. Conclusion: Although a down-regulation of A₁ adenosine receptor density was demonstrated after receptor agonist perfusion with normal flow, adenosine did not affect the density or functional activity of cardiac A₁ adenosine receptors in the ischemic myocardium. In contrast, muscarinic cholinoceptor density and function was down-regulated after prolonged ischemia. The lack of an agonist-induced down-regulation of A₁ adenosine receptors in the presence of decreasing activity of m-cholinoceptors suggests a growing importance of the adenosine system in myocardial ischemia.     

Affinity chromatography of A1 adenosine receptors of rat brain membranes

The A1 adenosine receptor of rat brain membranes has been solubilized with digitonin and purified approximately 150-fold by affinity chromatography. The digitonin-solubilized receptor, which can be labeled with 8-cyclopentyl-1,3-[3H]dipropylxanthine([3H]DPCPX), was adsorbed on xanthine amine congener (XAC)-linked agarose. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Adenosine agents blocked adsorption of solubilized receptor activity to the XAC-agarose with the appropriate A1 adenosine selectivity. For agonists, 8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than CV-1808, whereas, for antagonists, 8-cyclopentyltheophylline (CPT) greater than XAC greater than isobutylmethylxanthine = theophylline. The same A1 adenosine receptor specificity was observed for elution of [3H]DPCPX binding activity from the gel. XAC-agarose adsorbed 65-80% of the solubilized [3H]DPCPX binding activity and, after the gel was washed, 30-40% of the adsorbed activity could be eluted with 100 microM CPT, with specific binding activity of approximately 60 pmol/mg of protein. The order of potency of adenosine agonists [8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than (S)-phenylisopropyladenosine] and antagonists (DPCPX greater than XAC greater than CPT greater than isobutylmethylxanthine) with the affinity-purified preparation was found to be similar to that of the solubilized adenosine A1 receptor. This affinity chromatography procedure should prove to be valuable in the isolation and molecular characterization of A1 adenosine receptors.     

Involvement of adenosine A1 receptors in upregulation of nitric oxide by acyclic nucleotide analogues

Acyclic nucleoside phosphonates are a novel class of virostatics effective against replication of both DNA-viruses and retroviruses. They are synthetic analogues of natural nucleotide monophosphates, and purine derivatives thus represent counterparts of AMP. Mono- and di-phosphorylated species are analogues of natural ADP and ATP, respectively. A number of these compounds are endowed with immunostimulatory and immunomodulatory potential. We investigated whether their augmenting effect on the interferon-gamma-primed production of nitric oxide (NO) by murine macrophages is mediated by purinoceptors. The test compounds comprise alterations at the N(6)-group of the heterocyclic base, i.e., adenine or 2,6-diaminopurine, and at the N(9)-side chain, represented by 9-[2-(phosphonomethoxy)ethyl] and 9-[2-(phosphonomethoxy)propyl] moieties: 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir], N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-cyclopropyl-(R)-PMPDAP], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-cyclopentyl-(R)-PMPDAP], N(6)-dimethylaminoethyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-dimethylaminoethyl-(R)-PMPDAP], N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-isobutyl-PMEDAP), N(6)-cyclopentyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclopentyl-PMEDAP), N(6)-cyclooctyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclooctyl-PMEDAP), and N(6)-cyclohexylmethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclohexylmethyl-PMEDAP). The cells were cultured in the presence of interferon-gamma (5000 pg/ml) and test compounds (2-50 microM). Formation of nitrites was determined after 24 h using Griess reagent. It was inhibited by specific and nonspecific antagonists of adenosine A(1) receptors (IC(50) for 8-cyclopentyl-1,3-dipropylxanthine [CPX] was approximately 10 microM), while all other purine P(1) and purine P(2) receptor antagonists remained ineffective to suppress the NO-synergistic effect of acyclic nucleoside phosphonates.     

Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine: involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.     

Differential expression of renal adenosine A(1) receptors induced by acute renal failure

The distribution of renal adenosine A(1) receptors was investigated in rats with glycerol- or mercuric chloride (HgCl(2))-induced acute renal failure. Receptors were localised by autoradiography using [(3)H]8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX), a selective A(1) adenosine receptor antagonist. In saline-injected control animals, significant labelling with [(3)H]DPCPX was detected in glomeruli, the inner stripe of outer medulla, and the inner medulla. Sixteen hours following induction of glycerol-induced acute renal failure (ARF), a 34% increase in labelling in glomeruli was noted compared to saline-injected controls, and by 48 hr, glomerular labelling had increased by 200%. In addition, 48 hr following glycerol injection, significant labelling was now detected in the cortical labyrinth and medullary rays whilst, in the inner medulla, labelling had decreased by 34%. By contrast to glycerol-induced ARF, the only significant change noted 48 hr following induction of HgCl(2)-induced ARF was a 39% decrease in labelling in the inner medulla. It is concluded that glycerol-induced ARF results in differential expression of renal adenosine A(1) receptors with increased expression in the cortex and reduced expression in the inner medulla. Increased density of A(1) receptors in glomeruli may account, at least in part, for the increased renal vasoconstrictor response to adenosine and depressed glomerular filtration rate noted previously in this type of acute renal failure.