Human target validation of phosphoinositide 3-kinase (PI3K)ß: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kß inhibitor.

BACKGROUND: Based on in vitro and animal data, PI3Kß is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the novel, short-acting inhibitor AZD6482. METHODS AND RESULTS: AZD6482 is a potent, selective and ATP competitive PI3Kß inhibitor (IC(50) 0.01 µm). A maximal anti-platelet effect was achieved at 1 µm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti-thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3-h infusion. The ex vivo anti-platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin-induced human adipocyte glucose uptake in vitro (IC(50) of 4.4 µm). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 µm but reduced by about 60% at a plasma exposure of 27 µm. In man, the homeostasis model analysis (HOMA) index increased by about 10-20% at the highest plasma concentration of 5.3 µm. CONCLUSIONS: This is the first human target validation for PI3Kß inhibition as anti-platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at 'supratherapeutic' plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.

Cell-based multiwell assays for the detection of substrate accumulation and oxidation.

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping (14)CO(2) produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling.

A 68930 and dihydrexidine inhibit locomotor activity and d-amphetamine-induced hyperactivity in rats: a role of inhibitory dopamine D(1/5) receptors in the prefrontal cortex?

The behavioral and biochemical effects of the full dopamine D(1/5) receptor agonists, dihydrexidine and (1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl (A 68930), were examined in rats. Both A 68930 (0-4.6 mg kg(-1), s.c.) and dihydrexidine (0-8.0 mg kg(-1), s.c.) caused a dose-dependent suppression of locomotor activity, as assessed in an open-field. This locomotor suppression was dose-dependently antagonized by the selective dopamine D(1/5) receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine HCl (SCH 23390; 0-5.0 microg kg(-1), s.c.), but not by the selective dopamine D(2/3) receptor antagonist raclopride (0-25.0 microg kg(-1), s.c.). Furthermore, A 68930 and dihydrexidine did not cause any locomotor activity in habituated rats that displayed a very low base-line activity. Neither did A 68930 nor dihydrexidine produce any excessive stereotypies that could possibly interfere with and mask ambulatory activity. In fact, both A 68930 and dihydrexidine potently blocked hyperactivity produced by d-amphetamine (0-4.0 mg kg(-1), s.c.). Such findings traditionally would be interpreted as a sign of potential antipsychotic properties of A 68930 and dihydrexidine. Examination of neuronal activation, as indexed by the immediate early gene c-fos, showed that A 68930 and dihydrexidine caused a highly significant expression of c-fos in the medial prefrontal cortex. This c-fos expression was sensitive to treatment with SCH 23390, but not with raclopride. The effects of A 68930 and dihydrexidine on c-fos expression in caudate putamen or nucleus accumbens were less marked, or undetectable. The results indicate that stimulation of dopamine D(1/5) receptors, possibly in the medial prefrontal cortex, is associated with inhibitory actions on locomotor activity and d-amphetamine-induced hyperactivity. Assuming an important role of prefrontal dopamine D(1/5) receptors in schizophrenia, such inhibitory actions of dopamine D(1/5) receptor stimulation on psychomotor activation may have interesting clinical implications in the treatment of schizophrenia.

Lack of efficacy of 'naked' small interfering RNA applied directly to rat brain.

UNLABELLED: The intrastriatal infusions of 'naked' small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0-10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed. CONCLUSION: The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.

Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition.

Adenosine tonically inhibits synaptic transmission through actions at A(1) receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A(2A) receptor activation or from indirect control of inhibitory GABAergic transmission. The A(2A) receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA(A) and GABA(B) receptor antagonists, picrotoxin (50 microM) and CGP 55845 (1 microM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A(2A) receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A(1) receptors with N(6)-cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K(+)-evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 microM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 microM) and bisindolylmaleimide (1 microM), but not by the protein kinase A inhibitor, H-89 (1 microM). Isoproterenol (30 microM), but not CGS 21680 (10-300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A(1) receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([(3)H]DPCPX, 0.5 nM) with an EC(50) of 1 microM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A(1) receptors. Our results suggest that A(2A) receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A(1) receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A(2A) receptors to allow frequency-dependent plasticity phenomena without compromising the A(1) receptor-mediated neuroprotective role of adenosine.

Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells.

The potency of adenosine and inosine as agonists at human adenosine receptors was examined in a functional assay using changes in cyclic AMP (cAMP) formation in intact Chinese hamster ovary (CHO) cells stably transfected with the human A1, A2A, A2B, and A3 receptors. Adenosine increased cAMP formation in cells expressing the A2A (EC(50): 0.7 microM) and A2B (EC(50): 24 microM) receptors and inhibited forskolin (0.3-3 microM)-stimulated cAMP formation in cells expressing the A1 (EC(50): 0.31 microM) and A3 receptors (EC(50): 0.29 microM). The potency of adenosine at the A2A and A2B receptors was not altered by the presence of the uptake inhibitor nitrobenzylthioinosine (NBMPR), whereas it was increased about 6-fold by NBMPR at the A1 and A3 receptors. In the presence of NBMPR, inosine was a potent agonist (EC(50): 7 and 0.08 microM at the A1 and A3 receptors, respectively), but with low efficacy especially at the A3 receptors. No effect of inosine was seen at the A(2) receptors. Caffeine, theophylline, and paraxanthine shifted the dose-response curve for adenosine at the A1, A2A, and A2B receptors. These results indicate that adenosine is the endogenous agonist at all human adenosine receptors and that physiological levels of this nucleoside can activate A1, A2A, and A3 receptors on cells where they are abundantly expressed, whereas pathophysiological conditions are required to stimulate A2B receptors to produce cyclic AMP.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Structure and function of adenosine receptors and their genes.

Four adenosine receptors have been cloned from many mammalian and some non-mammalian species. In each case the translated part of the receptor is encoded by two separate exons. Two separate promoters regulate the A1 receptor expression, and a similar situation may pertain also for the other receptors. The receptors are expressed in a cell and tissue specific manner, even though A1 and A2B receptors are found in many different cell types. Emerging data indicate that the receptor protein is targeted to specific parts of the cell. A1 and A3 receptors activate the Gi family of G proteins, whereas A2A and A2B receptors activate the Gs family. However, other G proteins can also be activated even though the physiological significance of this is unknown. Following the activation of G proteins several cellular effector pathways can be affected. Signaling via adenosine receptors is also known to interact in functionally important ways with signaling initiated via other receptors.

Adenosine A(2A) receptors are colocalized with and activate g(olf) in rat striatum.

In situ hybridization with cRNA probes showed A(2A) receptor and G(olf) mRNAs to be abundantly expressed in caudate putamen, nucleus accumbens, and olfactory tubercle, whereas G(s) mRNA shows a comparatively low expression in regions expressing A(2A) receptors. In caudate putamen, 49% of the medium-sized neuron-like cells exhibited a strong signal for adenosine A(2A) receptor mRNA, and 98% showed a strong signal for G(olf) mRNA. In contrast, G(s) mRNA was found in only 12% of the medium-sized neuron-like cells in caudate putamen. The coexpression of adenosine A(2A) receptor mRNA with that of G(olf) or G(s) mRNAs was studied with double in situ hybridization. A large majority (91-95%) of the neurons in caudate-putamen that contained adenosine A(2A) receptor mRNA also expressed G(olf) mRNA, whereas only 3 to 5% of the neurons with adenosine A(2A) receptor mRNA coexpressed G(s) mRNA. The A(2A) receptor agonist CGS 21680 [2-[p-(2-carbonylethyl)phenylethylamino-5'-N-ethylcarboxa midoadenosin e] dose dependently activated G(olf) subunits in striatal membranes as shown by photolabeling with [alpha-(32)P]m-acetylanilido-GTP followed by immunoprecipitation with a specific antibody against G(olf). Transfection of G(olf) cDNA into Chinese hamster ovary cells, which stably express human adenosine A(2A) receptors, led to an increased efficacy of CGS 21680, as evidenced by a stronger cAMP response, indicating that activation of G(olf) by A(2A) receptors leads to a biological signal. In conclusion, these results provide anatomical and biochemical evidence that adenosine A(2A) receptors stimulate G(olf) rather than G(s) in striatum.

GTP differentially affects antagonist radioligand binding to adenosine A(1) and A(2A) receptors in human brain.

The effect of guanosine triphosphate (GTP) on the interaction of antagonists with human adenosine A(1) and A(2A) receptors was studied using whole-hemisphere sections from human brain and membranes from Chinese hamster ovary (CHO) cells expressing human A(1) and A(2A) receptors. Adenosine A(1) receptors, studied using [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as radioligand, showed the expected regional distribution in human brain. Addition of 500 microM GTP significantly increased (23-55%) [3H]DPCPX binding in all regions measured. In CHO cells transfected with human adenosine A(1) receptor cDNA, the number of receptors, B(max), increased from 401 (359-442) to 667 (592-743) fmol/mg protein upon addition of GTP. [3H]5-Amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2, 4-triazolo-[1,5-c]-pyrimidine ([3H]SCH 58261), a selective adenosine A(2A) receptor ligand, showed saturable binding to membranes from CHO cells transfected with adenosine A(2A) receptor cDNA and was localized to striatum and globus pallidus in human brain sections. Addition of GTP did not significantly change [3H]SCH 58261 binding to brain sections or CHO cell membranes. These results indicate that human A(1) and A(2A) receptors are not substantially different from those of the rat as regards regulation by GTP and interactions with endogenous adenosine in binding experiments. However, the relative abundance of the receptors differs between species, and this may be related to the differences observed in the potency of the endogenous agonist.

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Reciprocal interactions between adenosine A2A and dopamine D2 receptors in Chinese hamster ovary cells co-transfected with the two receptors.

Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal gamma-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPgammaS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose-response curve for quinpirole on adenosine 3',5'-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPgammaS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in EC50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.

Signaling via A2A adenosine receptor in four PC12 cell clones.

PC12 cells are genetically labile and so-called wild-type cells comprise multiple subclones. We have examined the A2A adenosine receptor signal transduction pathways in four such clones (denoted clones 1, 19, 21 and 27) of PC12 cells. Adenosine A2A, A2B and A1 receptor mRNAs were detected in all four clones by RT-PCR, whereas no A3 receptor mRNA was found. A2A receptors were quantitated by radioligand binding using the antagonist radioligand [3H]SCH 58261 ([3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine). The Bmax was highest in clone 1 followed by clones 21, 19 and 27. Whereas the amount of G(i) protein appeared similar in all four clones, the amount of G(s) protein was higher in clones 21 and 27 than in the other two clones. Maximal responses to the non-selective adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) were similar to those observed with the selective adenosine A2A receptor agonist CGS 21680 (2-[p-(2-carbonylethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine), and were approximately equal in clones I and 21, but lower in clone 19 and very low in clone 27. For both compounds EC50 was significantly higher in clone 27 than in clone 1. In both clones the response to NECA could be competitively antagonized by a selective adenosine A2A antagonist, SCH 58261. The present results show that different clones of PC 12 cells differ widely in the cAMP increase induced by adenosine analogues and that this is due to differences in the amount of adenosine A2A receptor, G protein and effector. A large difference in receptor number resulted in differences in potency of an agonist.

Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors.

To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.

Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells.

Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.

Cellular expression of adenosine A2A receptor messenger RNA in the rat central nervous system with special reference to dopamine innervated areas.

The cellular distribution of adenosine A2A receptor messenger RNA in the central nervous system was investigated using in situ hybridization with ribonucleotide probes. A specific expression was found in the dorsal (i.e. caudate putamen) and ventral (i.e. nucleus accumbens and olfactory tubercle) striatum, the lateral septum and in some cerebellar Purkinje cells. Simultaneous detection of radioactive and non-radioactive probes showed that the majority of adenosine A2A receptor messenger RNA-containing neurons in the dorsal and ventral striatum co-expressed dopamine D2 receptor messenger RNA and preproenkephalin A messenger RNA. However, a minor sub-population of neurons expressing adenosine A2A receptor messenger RNA, but not preproenkephalin A messenger RNA, was found in clusters along the ventral border of the nucleus accumbens. Only a small number of striatal neurons expressing dopamine D1 receptor or substance P messenger RNAs also expressed adenosine A2A receptor messenger RNA. Finally, in the ventral part of nucleus accumbens and in the olfactory tubercle a major sub-population of neurons expressed preproenkephalin A messenger RNA, but not adenosine A2A receptor messenger RNA. Cholinergic interneurons did not express adenosine A2A receptor messenger RNA. Thus, the extensive co-localization of adenosine A2A and dopamine D2 receptors previously described in the dorsal striatum extends into its ventral part. There is also a high degree of co-expression of adenosine A2A receptor messenger RNA and preproenkephalin A messenger RNA in the ventral striatum, but within this region several topologically defined sub-populations of neurons express only one of these transcripts. A majority of the adenosine A2A receptor messenger RNA-containing neurons in the lateral septum did contain preproenkephalin A messenger RNA, whereas only a few co-expressed dopamine D2 receptor messenger RNA. This detailed investigation demonstrates that most of the subcortical areas innervated by dopamine have an abundant, although restricted expression of the adenosine A2A receptor gene and that this receptor is expressed in very few cells outside these areas. These results predict that adenosine A2A receptors are involved not only in motor behaviour, but also in goal-oriented behaviours.

Characterization of human A2A adenosine receptors with the antagonist radioligand [3H]-SCH 58261.

1. We have characterized the binding of the new potent and selective antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o[1,5- c]pyrimidine, [3H]-SCH 58261, to human cloned A2A adenosine receptors. 2. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]-SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (KD = 2.3 nM) and limited capacity (apparent Bmax = 526 fmol mg-1 protein). 3. Competition experiments revealed that binding of 0.5 nM [3H]-SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) = 2-phenylaminoadenosine (CV 1808) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > R-N6-phenylisopropyladenosine (R-PIA) > or = N6-cyclohexyladenosine (CHA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine receptor antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > SCH 58261 > xanthine amine congener (XAC) > (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3- dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > theophylline. 5. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. 6. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]-CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.

In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants.

A full-length cDNA copy (PLRVfl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA. In addition, PLRVfl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation. Both transient and stable expression of PLRVfl resulted in the synthesis of genomic and subgenomic PLRV RNAs. Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection. This is the first example of the constitutive expression of a phloem-limited virus in planta.

Localization of adenosine receptor messenger RNAs in the rat eye.

Adenosine is present in all cells and body fluids and has been suggested to play several roles in the physiology of ocular tissues. The present study was undertaken to determine which types of adenosine receptor mRNAs are present in the rat eye, and where they are expressed. RNA or deoxyoligodeoxynucleotides complementary to rat adenosine receptor subtypes A1, A2A, A2B and A3 were used to generate 35S labeled antisense and sense probes. The probes were then used for in situ hybridization on 10 microm cryosections of the rat eye including the cornea, iris, ciliary body, lens, retina, choroid and sclera. A1, A2A and A2B receptor mRNAs were demonstrated in the ciliary processes. A1 receptor mRNA was also expressed in the ganglion cell layer of the retina. The retina also showed A2A receptor mRNA expression, which was most prominent in the inner nuclear layer and less prominent in the ganglion cell layer and outer nuclear layer. Weak A2A expression was found in the retinal pigment epithelium and choriocapillaris. No significant expression of A3 receptor mRNA was found in the rat eye. In conclusion, using in situ hybridization, we have demonstrated expression of mRNA for A1, A2A and A2B adenosine receptors in the rat eye. The expression patterns support specific roles for adenosine in the ciliary process and retina.

Adenosine A2A receptors modulate the binding characteristics of dopamine D2 receptors in stably cotransfected fibroblast cells.

In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.