Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity

A series of N6-bicyclic and N6-(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N6-adamantyl substitution in combination with 5′-N-ethylcarboxamido or 5′-hydroxymethyl groups. In addition, we determined that 5′-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compounds might have therapeutic potential.

Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of pesticides on the natural enemies of the different agrosystems. A part of this work has been focusses on evaluating the direct and indirect effects of kaolin particle films and two copper-based products (Bordeaux mixture and copper oxychloride) through different laboratory, extended laboratory and semi-field experiments. Two natural enemies have been chosen: Psyttalia concolor (Szèpligeti), a parasitoid of the olive fruit fly, and Chilocorus nigritus (F.), predator of Diaspididae. This predator has been used instead of C. bipustulatus (L.), which is the species found in olive orchards. Kaolin mainly acts as a repellent of insects and/or as an oviposition deterrent. It is used in olive groves to control the olive fruit fly and the olive moth. Copper is applied against fungal and bacterial diseases. In olive groves it is used against the olive leaf spot and other diseases. No statistical differences were found in any of the experiments performed, compared to the controls, except when the oral toxicity of the products was evaluated on P. concolor females. In this case, kaolin and copper oxychloride caused a higher mortality 72 hours after the treatments, and both kaolin and the two copper formulations decreased females’ life span. Reproductive parameters were only negatively affected when kaolin was ingested. Apart from these experiments, due to the uncommon mode of action of kaolin, two extra experiments were carried out: a dual choice and a no-choice experiment. In this case, both P. concolor females and C. nigritus adults showed a clear preference for the untreated surfaces when they had the possibility of choosing between a treated surface and an untreated one. When there was no choice, no statistical differences were found between the treatments and the controls. Furthermore, the efficacy and the selectivity of three insect growth regulators (methoxyfenozide, tebufenozide and RH-5849) on B. oleae and P. concolor, respectively, have also been evaluated. In addition to laboratory experiments to evaluate the toxicity of the insecticides, also molecular approaches were used. RNA of both insects was isolated. cDNA was subsequently synthesized and the complete sequences of the ligand biding domain (LBD) of the ecdysone receptor of each insect were then determined. Afterwards the three dimensional structures of both LBDs were constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the LBD was performed. Both toxicity assays and molecular docking approaches showed that either methoxyfenozide or tebufenozide had no negative effects nor on B. oleae nor on P. concolor. In contrast, RH-5849 had no deleterious effect to the parasitoid but decreased olive fruit fly adults’ life span, especially when they were in contact with the fresh residue of the insecticide applied on a glass surface. The docking study of RH-5849 molecule has shown a very light hindrance with the wall of the LBD pocket. This means that this molecule could more or less adjust in the cavity. Thus, searching of new insecticides for controlling the olive fruit fly could be based on the basic lead structure of RH-5849 molecule.

Nonsteroidal moulting hormone agonists: effects on protein synthesis and cuticle formation in colorado potato beetle larvae.

Larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say), that were orally treated with RH-0345 at 0.1 mg l?1, RH-5849 at 10 and 50 mg l?1, tebufenozide at 2 g l?1, and 20-hydroxyecdysone at 2 g l?1, showed symptoms of prematuremoulting, followed by inhibition of ecdysis. In addition, fresh weight gain and total protein content were blocked. The effects on haemolymphal and cuticular polypeptides after PAGE were linked with premature, new epicuticle deposition as was observed under the electron microscope. These observations support the concept that the ecdysteroid-mimicking action of the three nonsteroidal molecules is specific

Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-XL

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVM1 model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and prodopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-XL induces an increase in the expression of key developmental genes (MSX1, NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (EN1, LMX1B, NURR1 and PITX3). As a result, Bcl-XL anticipates and enhances DAn generation.

Dopamine receptor 4 promoter polymorphism modulates memory and neuronal responses to salience

Animal models and human functional imaging data implicate the dopamine system in mediating enhanced encoding of novel stimuli into human memory. A separate line of investigation suggests an association between a functional polymorphism in the promoter region for the human dopamine 4 receptor gene (DRD4) and sensitivity to novelty. We demonstrate, in two independent samples, that the -521Cmayor queT DRD4 promoter polymorphism determines the magnitude of human memory enhancement for contextually novel, perceptual oddball stimuli in an allele dose-dependent manner. The genotype-dependent memory enhancement conferred by the C allele is associated with increased neuronal responses during successful encoding of perceptual oddballs in the ventral striatum, an effect which is again allele dose-dependent. Furthermore, with repeated presentations of oddball stimuli, this memory advantage decreases, an effect mirrored by adaptation of activation in the hippocampus and substantia nigra/ventral tegmental area in C carriers only. Thus, a dynamic modulation of human memory enhancement for perceptually salient stimuli is associated with activation of a dopaminergic-hippocampal system, which is critically dependent on a functional polymorphism in the DRD4 promoter region.

Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Prominence of the dopamine D2 short isoform in dopaminergic pathways

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.

Dopamine-dependent responses to morphine depend on glucocorticoid receptors

Previous work has shown that glucocorticoid hormones facilitate the behavioral and dopaminergic effects of morphine. In this study we examined the possible role in these effects of the two central corticosteroid receptor types: mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). To accomplish this, specific antagonists of these receptors were infused intracerebroventricularly and 2 hr later we measured: (i) locomotor activity induced by a systemic injection of morphine (2 mg/kg); (ii) locomotor activity induced by an infusion of morphine (1 μg per side) into the ventral tegmental area, which is a dopamine-dependent behavioral response to morphine; (iii) morphine-induced dopamine release in the nucleus accumbens, a dopaminergic projection site mediating the locomotor and reinforcing effects of drugs of abuse. Blockade of MRs by spironolactone had no significant effects on locomotion induced by systemic morphine. In contrast, blockade of GRs by either RU38486 or RU39305, which is devoid of antiprogesterone effects, reduced the locomotor response to morphine, and this effect was dose dependent. GR antagonists also reduced the locomotor response to intraventral tegmental area morphine as well as the basal and morphine-induced increase in accumbens dopamine, as measured by microdialysis in freely moving rats. In contrast, spironolactone did not modify dopamine release. In conclusion, glucocorticoids, via GRs, facilitate the dopamine-dependent behavioral effects of morphine, probably by facilitating dopamine release. The possibility of decreasing the behavioral and dopaminergic effects of opioids by an acute administration of GR antagonists may open new therapeutic strategies for treatment of drug addiction.

Dopamine Autoreceptor Regulation of a Hypothalamic Dopaminergic Network

Open Access funded by European Research Council Acknowledgments The authors thank Drs. Gilberto Fisone, Jessica Ausborn, Abdel El Manira, Gilad Silberberg, and members of the C.B. laboratory for advice, as well as Paul Williams for expert help with the graphical abstract. This study was supported by a Starting Investigator Grant from the ERC (ENDOSWITCH 261286), the Swedish Research Council (2010-3250), Novo Nordisk Fonden, and the Strategic Research Programme in Diabetes at Karolinska Institutet.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Receptors induce chemotaxis by releasing the βγ subunit of Gi, not by activating Gq or Gs

Many chemoattractants cause chemotaxis of leukocytes by stimulating a structurally distinct class of G protein-coupled receptors. To identify receptor functions required for chemotaxis, we studied chemotaxis in HEK293 cells transfected with receptors for nonchemokine ligands or for interleukin 8 (IL-8), a classical chemokine. In gradients of the appropriate agonist, three nonchemokine Gi-coupled receptors (the D2 dopamine receptor and opioid μ and δ receptors) mediated chemotaxis; the β2-adrenoreceptor and the M3-muscarinic receptor, which couple respectively to Gs and Gq, did not mediate chemotaxis. A mutation deleting 31 C-terminal amino acids from the IL-8 receptor type B quantitatively impaired chemotaxis and agonist-induced receptor internalization, but not inhibition of adenylyl cyclase or stimulation of mitogen-activated protein kinase. To probe the possible relation between receptor internalization and chemotaxis, we used two agonists of the μ-opioid receptor. Morphine and etorphine elicited quantitatively similar chemotaxis, but only etorphine induced receptor internalization. Overexpression of two βγ sequestering proteins (βARK-ct and αt) prevented IL-8 receptor type B-mediated chemotaxis but did not affect inhibition of adenylyl cyclase by IL-8. We conclude that: (i) Nonchemokine Gi-coupled receptors can mediate chemotaxis. (ii) Gi activation is necessary but probably not sufficient for chemotaxis. (iii) Chemotaxis does not require receptor internalization. (iv) Chemotaxis requires the release of free βγ subunits.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+ channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type α1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α1A subunit. The resulting chimeric α1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type α1C subunit and 92% of the binding energy of the l-type α1S subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca2+ channels containing α1A/DHPS was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of α1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.