Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.     

The P2Y(1) receptor closes the N-type Ca(2+) channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists

The rat P2Y(1) nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro-injection of the receptor cRNA or cDNA. ADP inhibited the N-type Ca(2+) current by 64%, with EC(50) 8.2 nM, an action blocked competitively by the P2Y(1) receptor antagonist adenosine 3', 5'-bis-phosphate (K(i) 0.7 microM). 2-Methylthio-ADP inhibited the Ca(2+) current likewise, but with EC(50) 0.57 nM, giving the highest potency reported therewith for P2Y(1). Significantly, ATP and 2-methylthio-ATP were also agonists, the latter again at a very high potency (EC(50) 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N-type Ca(2+) currents and hence can reduce transmitter release or increase neuronal excitability.     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y(1) receptor

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.     

Novel modified adenosine 5'-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y(1) receptor: Biotinylated analogue potentially suitable for specific P2Y(1) receptor isolation

Rat brain P2Y(1) (rP2Y(1)) receptor-transfected human embryonic kidney cells (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (V?hringer C, Sch?fer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for further studying novel adenine nucleotide analogues (Halbfinger et al. J Med Chem 1999;42:5325-37) and for the biochemical characterization of the P2Y(1) receptor. By measurement of intracellular Ca(2+) release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC(50) values of 1.3, 5, and 60 nM were determined, markedly lower than the value for ATP (130 nM). The EC(50) for 2-BuSADP was 1.1 nM. The corresponding 8-substituted ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuSATP > 8-BuNHATP approximately 8-BuOATP). AMP induced intracellular Ca(2+) release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC(50) = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC(50) = 6 nM), revealing that even more bulky substituents linked to the C-2 position bind with high affinity at the P2Y(1) receptor. This biotinylated probe was successfully used for the enrichment of the P2Y(1) receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y(1) receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y(1) receptor represent a powerful model system for pharmacological characterization of the P2Y(1) receptor, circumventing problems associated with natural systems. They provide a means for the development of P2Y(1) ligands of high potency and a good source for obtaining purified P2Y(1) receptor.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.     

Binding of adenosine receptor ligands to brain of adenosine receptor knock-out mice: evidence that CGS 21680 binds to A1 receptors in hippocampus

The adenosine receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5-N-ethylcarboxamidoadenosine (CGS 21680) is generally considered to be a selective adenosine A_2A receptor ligand. However, the compound has previously been shown to exhibit binding characteristics that are not compatible with adenosine A_2A receptor binding, at least in brain regions other than the striatum. We have examined binding of [³H]CGS 21680 and of antagonist radioligands with high selectivity for adenosine A₁ or A_2A receptors to hippocampus and striatum of mice lacking either adenosine A₁ (A1R^(–/–)) or A_2A (A2AR^(–/–)) receptors. Both receptor autoradiography and membrane binding techniques were used for this purpose and gave similar results. There were no significant changes in the binding of the A₁ receptor antagonist [³H]DPCPX in mice lacking A_2A receptors, or in the binding of the A_2A receptor antagonists [³H]SCH 58261 and [³H]ZM 241385 in mice lacking A₁ receptors. Furthermore, [³H]CGS 21680 binding in striatum was abolished in the A2AR^(–/–), and essentially unaffected in striatum from mice lacking A₁ receptors. In hippocampus, however, binding of [³H]CGS 21680 remained in the A2AR^(–/–), whereas binding was virtually abolished in the A1R^(–/–). There were no adaptive alterations in A_2A receptor expression in this region in A1R^(–/–) mice. Thus, most of the [³H]CGS 21680 binding in hippocampus is dependent on the presence of adenosine A₁ receptors, but not on A_2A receptors, indicating a novel binding site or novel binding mode.     

Acyclic and cyclopropyl analogues of adenosine bisphosphate antagonists of the P2Y1 receptor: structure-activity relationships and receptor docking.

The activation of P2Y1 receptors in platelets contributes to platelet aggregation, and selective antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J. Med. Chem. 2000, 43, 746-755) that acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine, are moderately potent P2Y1 receptor antagonists. In this study we have varied the chain structure, to include asymmetric substitution, olefinic, and cyclopropyl groups. These antagonists inhibited the stimulation of phospholipase C in turkey erythrocyte membranes induced by 30 nM 2-MeS-ADP in the micromolar range. In the series of symmetrically branched aliphatic groups substituted with two phosphate groups, the optimal antagonist potency occurred with the 2-methylpropyl group. A 2-chloro-N(6)-methyladenine derivative, 2-[2-(2-chloro-6-methylaminopurin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate) (7), was a full antagonist at the P2Y1 receptor with an IC(50) value of 0.48 microM. Esterification of one of the phosphate groups or substitution with O-acetyl greatly reduced the antagonist potency at the P2Y1 receptor. Removal of a methylene group of 7 or inclusion of an olefinic or cyclopropyl group also reduced potency. A pair of enantiomeric glycerol derivatives demonstrated a 5-fold stereoselectivity for the S-isomer. Stereoisomerically defined analogues of 7 containing a cyclopropyl group in place of the branched carbon were less potent than 7 as antagonists, with IC(50) values of 2-3 microM. No agonist activity was observed for these analogues. A new rhodopsin-based molecular model of the P2Y1 receptor indicated that the optimal docked orientation of the two monophosphate moieties relative to the adenine N(6) (compared to a rigid, bicyclic analogue) was consistent with the dependence of antagonist potency on chain length. The 3'-phosphate was predicted to occupy a restricted space, deeper in the binding cleft than the 5'-phosphate location. In summary, modification of the flexible spacer chain linking bisphosphate groups to the adenine moiety provided many moderately potent antagonists.     

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5'-O-(1-boranotriphosphate) derivatives

P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-alpha-B) were synthesized by substitution of a nonbridging O at P(alpha) with a BH(3) group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y(1) and rat P2Y(2) receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y(1)-GFP, rP2Y(2)-GFP). We investigated agonist-induced receptor endocytosis, [Ca(2+)](i) rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y(1)-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-alpha-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca(2+) release by these compounds in HEK 293 cells stably transfected with rP2Y(1). In case of rP2Y(2)-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca(2+) release, AA release and in inducing receptor endocytosis. The different ATP-alpha-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y(1) receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes.     

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y₄ (hP2Y₄) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y₄ receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2-MeSATP=Ap₄A>UDP(pure). ADP, ATPγS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y₄ is more like the P2U receptor subtype. The rP2Y₄ receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y₂ receptor (a known P2U receptor). Thus, rP2Y₄ and the P2Y₂ subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.     

Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells.

The aim of this study was to investigate the stimulating effects on arachidonic acid release of P2Y1 and P2Y2 receptor-selective agonists, 2-methylthio-ATP (2MeSATP) and UTP, respectively, in bovine pulmonary artery endothelial cells. Exposure of cells to 2MeSATP and UTP led to the release of arachidonic acid, a response which was abolished by the removal of extracellular Ca2+ and methyl arachidonyl fluorophosphonate. Phorbol 12-myristate 13-acetate (PMA) itself not only stimulated arachidonic acid release but also played a permissive role in the response to UTP. However, PMA failed to enhance the arachidonic acid response induced by 2MeSATP, probably due to greater attenuation of the [Ca2+]i increase caused by 2MeSATP than UTP. Inhibition of protein kinase C with Ro 31-8220 (1-[3-(amidinothio) propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide -methane sulphate) and staurosporine, but not with Go 6976 (12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-indolo(2, 3-a)pyrrolo(3,4-c)carbazole), reduced the arachidonic acid response of 2MeSATP, UTP and PMA. PMA-induced potentiation of the UTP response reached a maximum after a 1-h preincubation, then declined and eventually lost its effect when the preincubation lasted up to 8 h. Among the protein kinase C isoforms present in endothelial cells, betaI and epsilon could be down-regulated by treatment with PMA for 4-24 h. PD 098059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) inhibited extracellular signal-regulated protein kinase activation, cytosolic phospholipase A2 phosphorylation and arachidonic acid release caused by 2MeSATP, UTP and PMA. Taken together, our results demonstrate that P2Y1 and P2Y2 purinoceptors mediate arachidonic acid release by activating cytosolic phospholipase A2 through an elevation of [Ca2+]i and protein kinase C epsilon-, extracellular signal-regulated protein kinase-dependent phosphorylation.     

Characterisation of P2Y(1)-like receptor in cultured rat pineal glands

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.     

Inhibition of platelet P2Y12 and alpha2A receptor signaling by cGMP-dependent protein kinase

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.     

A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleoside triphosphates

We recently cloned and expressed a novel P2Y receptor (tp2y receptor) from a turkey cDNA library. Expression of this receptor in 1321N1 human astrocytoma cells confers nucleotide-dependent stimulation of phospholipase C activity; however, as we demonstrate here, it also confers nucleotide-dependent inhibition of adenylyl cyclase. Both the phospholipase C and adenylyl cyclase responses were promoted by receptor agonists over a similar range of concentrations. Moreover, not only did UTP and ATP activate the avian receptor but ITP, GTP, xanthosine 5'-triphosphate, and CTP were also agonists, with EC(50) values ranging between 0.1 and 1 microM. Similar potencies, rank-order, and selectivity of nucleotide agonists were also demonstrated for intracellular Ca(2+) mobilization measured during a 30-s stimulation under constant superfusion conditions. This observation indicates that receptor activation by nucleoside 5'-triphosphates is not produced by interconversion of these nucleotides into ATP or UTP. Pretreatment of cells with pertussis toxin completely abolished the inhibitory effect of nucleotide agonists on adenylyl cyclase, whereas the activation of phospholipase C was only partially inhibited. These results demonstrate that the avian P2Y receptor is a nucleoside triphosphate receptor of broad agonist selectivity that interacts with both pertussis toxin-insensitive and -sensitive G proteins to activate phospholipase C and to inhibit adenylyl cyclase. This is the first cloned P2Y receptor that is clearly Gi/adenylyl cyclase-linked.     

Effects of uric acid and caffeine on A1 adenosine receptor binding in developing rat brain

Previous studies have demonstrated that elevated levels of serum uric acid or caffeine are associated with increased locomotor activity in rats and humans. Since uric acid and caffeine are structurally similar, it was hypothesized that these compounds alter locomotor activity through a common neural mechanism, specifically by acting as receptor antagonists at adenosine A1 binding sites. In vitro competition of caffeine and uric acid against the A1 agonist, [3H] cyclohexyladenosine ([3H]CHA), was conducted using homogenates of adult rat forebrain. Caffeine effectively competed for the A1 binding site as previously reported (IC50 = 107 microM), but uric acid failed to compete with [3H]CHA binding at concentrations within a relevant physiological range. Nevertheless, in vivo experiments indicated that chronic elevation of uric acid following allantoxanamide treatment of male rats on days 4-27 of life significantly decreased A1 receptor binding in the striatum, a region traditionally implicated in mammalian locomotion. In contrast, chronic caffeine treatment on days 4-27 of life caused an increase in A1 receptor binding in the cortex similar to increases reported previously in whole brain. These changes in A1 receptor binding following chronic elevation of uric acid or caffeine did not persist in rats that had been withdrawn from allantoxanamide or caffeine treatment for 14 days. Results from in vitro and in vivo experiments indicate that despite a similar molecular structure uric acid does not act by the same mechanism as caffeine to increase locomotor activity in rats.     

Effect of long term caffeine treatment on A1 and A2 adenosine receptor binding and on mRNA levels in rat brain

Summary The effect of long-term oral treatment with caffeine on A₁ and A₂ receptors in the rat brain was studied. Caffeine was added to the drinking water and the animals were sacrificed after a 12 day treatment period. The plasma caffeine concentration was close to 100 M. A₁ receptors were studied using quantitative autoradiography with [³H]cyclohexyladenosine (CHA). Caffeine treatment increased the number of A₁ receptors in the CA3 subfield of the hippocampus from 337 to 393 fmol/mg with no change in K_D (0.692 vs. 0.675 nM). A₁ mRNA was measured using Northern blots and quantitative in situ hybridization. There was no increase in A₁ mRNA. A_2a receptors, located in dopamine rich regions of the rat brain, were studied with quantitative autoradiography using [³H]CGS 21680 as the ligand, and the A_2a mRNA was determined using quantitative in situ hybridization. Caffeine treatment produced no significant change in either receptor number or mRNA, even though the apparent B_max tended to increase from 322±8 to 352±8 fmol/mg. The results show that treatment with caffeine in a dose that causes tolerance to several effects of caffeine and increases some effects of adenosine analogues increases the number of A₁ receptors without any change in A₁ mRNA, suggesting that the adaptive changes are at a post-translational level. There were no significant changes in A₂ receptors indicating that the two types are regulated differently and/or that the amount of endogenous agonist is sufficient to regulate A₁, but not A₂ receptors.Correspondence to B. Johansson at the above address